A fundamental feature of the pathogenesis of Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), is its ability to survive and grow in host macrophages. For more than five decades, many laboratories have investigated how Mtb interacts with and modulates the function of macrophages. Mtb is characterized by a ‘waxy’ coat, which confers its distinctive acid-fast staining properties. The complex cell envelope is important for pathogenesis and also allows Mtb to withstand adverse conditions (Dulberger et al., 2020). The mycobacterial envelope consists of a plasma membrane, peptidoglycan-arabinogalactan layer, outer membrane, and capsular layer (Dulberger et al., 2020). The outermost layers of the envelope are crucial in host-pathogen interactions given that they are directly able to interact with host cells. The capsule is composed primarily of a loose matrix of neutral polysaccharides (Kalscheuer et al., 2019), while the outer membrane is composed of long-chain mycolic fatty acids that are free, attached to trehalose, or covalently attached to the underlying arabinogalactan-peptidoglycan layer. The outer membrane also contains a complex array of unique lipids. Many of these lipids are bioactive; they can intercalate into host membranes, alter inflammatory signaling, disrupt phagosome maturation, and promote mycobacterial virulence (Cambier et al., 2020; Lerner et al., 2018; Quigley et al., 2017). Some outer membrane lipids, including phthiocerol dimycocerosate (PDIM), phenolic glycolipids, and sulfoglycolipids, are thought to act as antagonists of pathogen recognition receptors (PRRs) or to shield underlying pathogen associated molecular patterns (PAMPs) to prevent them from activating PRRs (Blanc et al., 2017; Cambier et al., 2014; Reed et al., 2004). Thus, the integrity of the envelope is crucial for host interactions and bacterial virulence.

Given the importance of Mtb-macrophage interactions, a mainstay of the experimental approach of many laboratories is the use of in vitro cultured Mtb to infect myeloid cells. However, the tendency of Mtb to form bacterial clumps has long presented an obstacle to these experiments, which depend on using precise and reproducible amounts of bacteria (Wells, 1946). For this reason, low concentrations of detergents are commonly added to culture media, but this does not fully resolve the problem. Therefore, additional measures are routinely taken to generate single cell suspensions, including sonicating, syringing, centrifuging, filtering, vortexing with glass beads, or some combination of these procedures. The use of these techniques varies widely across different laboratories, the methodology used is not always reported, and there has been little consideration as to how these techniques impact experimental outcomes.

We are interested in how mycobacterial protein and lipid effectors modulate macrophage responses. Sometimes our results differed from published data, leading us to question whether the method of preparing the bacilli explained the differences. However, there was minimal literature into how dispersing mycobacterial clumps impacts the envelope and host-pathogen interactions. Previous studies demonstrated that use of detergent and agitation can release capsular constituents (Lemassu et al., 1996; Sani et al., 2010). In addition, it was reported that Mtb that had been sonicated for 90 s were better able to bind to macrophages, and the bacterial envelope appeared uneven and bulging on transmission electron microscopy (Stokes et al., 2004). Another study showed that passing bacterial cultures through 5 μm pore filters improved reproducibility of high-throughput antibacterial drug screening compared to vortexing, but the impact on host-pathogen interactions was not assessed (Cheng et al., 2014). Given the lack of published studies addressing our concern, we compared three routinely used methods of preparing single cell suspensions of Mtb: low-speed spin, gentle sonication followed by low-speed spin, or filtration through a 5 μm filter. We found that the method of bacterial preparation had a marked impact on intracellular bacterial viability, the global transcriptional pattern of infected cells, macrophage secretion of key innate immune mediators, and the ultrastructure of the bacterial cell envelope. Finally, when comparing an Mtb mutant that lacks PDIM to WT bacilli, we found that the method of preparation had a substantial impact on the inflammatory response to the mutant bacilli.

More than 50 years ago, D’Arcy Hart demonstrated that M. tuberculosis avoids lysosomal delivery within macrophages (Armstrong and Hart, 1971). Ever since his landmark study, in an effort to understand fundamental mechanisms of TB pathogenesis, investigators have studied the interaction of Mtb with macrophages. They have also employed a variety of methods to disperse the bacilli to enable subsequent analysis. Unexpectedly, we found that two commonly used single cell preparation methods significantly impacted Mtb-host interactions: sonicated bacilli were hyperinflammatory, and 5μm-filtered Mtb were attenuated in macrophages. These effects were seen for H37Rv, HN878, and Erdman strains. In addition, we found that the method of preparation changes the impact of PDIM on the early macrophage transcriptional responses to Mtb. Consistent with the data of Hinman et al., 2021, who used a low-speed spin method to remove clumps, we found little impact of PDIM on the early TLR2-dependent response to centrifuged bacteria. However, if the bacteria were briefly sonicated then strains without PDIM elicited increased pro-inflammatory gene expression compared to control strains. This suggests that the mutant without PDIM is either more sensitive to sonication-induced damage or that it is more inflammatory once that damage occurs. It is important to point out that we only examined the early TLR2-dependent response. PDIM influences a variety of processes, including later TLR2-driven responses, phagosomal escape, and intracellular survival (Augenstreich et al., 2017; Barczak et al., 2017; Cambier et al., 2020; Hinman et al., 2021; Lerner et al., 2018; Osman et al., 2020; Quigley et al., 2017). It is possible that these other PDIM-dependent processes are not impacted by the preparation method. Nonetheless, our studies demonstrate that the preparation method needs to be considered in host-pathogen interaction studies, as it can change the interpretation of bacterial mutants and has a dramatic effect on TLR2-dependent responses and intracellular bacterial survival in bone marrow-derived macrophages.

Given the extensive use of macrophages in Mtb pathogenesis studies, there are surprisingly few studies investigating the impact of dispersal methods. A 2004 study demonstrated that prolonged sonication (5 min) reduces Mtb viability, while bacteria that had undergone gentle sonication (30 sec x 3) exhibited enhanced binding to macrophages and altered surface charge relative to syringed bacteria (Stokes et al., 2004). In that study, the sonicated bacteria had an altered cell envelope, which appeared uneven and bulging. Even though we sonicated for a shorter time (10 sec x 3), we also saw evidence of similar cell envelope disruption by TEM. In addition, we found that sonicated bacteria elicited orders-of-magnitude higher levels of TLR2-dependent transcriptional responses, leading to enhanced IL-1β and TNF-α secretion. This was mediated in part by material that was no longer cell associated, as even sterile-filtered samples activated macrophages. In addition, uninfected bystander cells that had been treated with so/sp preparations secreted TNF-α in the FluoroDOT assay. Our TEM findings suggest that sonication results in cell envelope damage and generation of small structures that resemble extracellular vesicles (EVs) that have been described in Mtb, although the vesicles that we saw are generally larger than the majority of EVs (Prados-Rosales et al., 2011). Mtb EVs are formed by an active process and contain immunomodulatory molecules including lipoarabinomannan and other TLR2 agonists (Athman et al., 2015; Palacios et al., 2021; Prados-Rosales et al., 2011). Whether the structures formed by sonication have similar content to EVs found in growing cultures will require further studies.

How might sonication and filtration lead to the distinct macrophage responses that we observed? Electron microscopy revealed that sonication and filtration cause different types of alteration to the cell envelope (Figure 7A). The cell envelope is an elaborately layered structure that contains a variety of lipid and protein PAMPs and virulence factors. Our data are consistent with a model in which PAMPs and virulence factors are differentially impacted by sonication and filtration (Figure 7B). In this model, sonication disrupts the cell envelope in a manner that makes PAMPs more highly exposed or accessible, while leaving the activity of virulence factors intact. In contrast, filtration (5μmF) disrupts the cell envelope in such a way that both virulence factors and PAMPs are inactivated and/or dispersed from the bacilli, rendering the bacteria both attenuated and less inflammatory. Bacteria that are subject only to low-speed spin are neither hyper-inflammatory nor attenuated, since both PAMPs and virulence factors are less perturbed. In the case in which the bacteria are sonicated and filtered, they are both hyper-inflammatory and attenuated, which can be explained by enhanced exposure of PAMPs through sonication and inactivation of virulence factors by filtration.

Figure 7

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This is one model that would explain our findings, but other scenarios could be envisioned. Mtb cultures are highly heterogenous, so we considered the possibility that a small minority of the so/sp bacilli were contributing to the heightened inflammatory response. However, the Fluoro-dot data, which allows us to visualize cytokine secretion at a single cell level, argue against this possibility. Similarly, if there were a small population of bacilli in the filtered sample that were inhibiting the inflammatory response, then, we would have expected the mixed samples to behave like the filtered sample, but rather we saw an intermediate phenotype. In terms of intracellular growth, there is undoubtedly heterogeneity within the population, with better intracellular growth on a population level in the so/sp and sp samples relative to filtration, with all samples having a mix of growth, stasis, and killing. Two aspects of heterogeneity that we assessed are clump size and capsule thickness. On a population level, we found a significant reduction in the thickness of the capsule of the 5μmF bacteria compared to both so/sp and sp bacteria. However, there was a wide distribution in cell envelope thickness, which may contribute to heterogeneity in macrophage responses to bacilli on a cell-to-cell level. Finally, we considered that differences in the degree of aggregation in the different bacterial preparations may account for differences in inflammatory potential or intracellular survival (Rodel et al., 2021), but heterogeneity in this aspect of the bacterial population was unlikely to explain the differences in outcomes. There may be other aspects of underlying heterogeneity in our samples that contribute to their distinct behavior or confound bulk measurements.

While our study was limited to three common single cell preparation methods, we expect that other techniques would also impact the mycobacterial envelope and host interactions. We queried PubMed for papers published in 2021 on Mtb and macrophages to determine which methods were commonly used (Supplementary file 3). Of the 119 papers, only 39.5% reported how they generated single cell suspensions. Of those that did report their methodology, 42.5% used more than one method. The most commonly reported methods were syringing, followed by sonication and low-speed centrifugation. Less often, filtering, vortexing with glass beads, or allowing gravity to sediment the larger clumps were used. Dispersing clumps with glass beads would likely disrupt the envelope, as studies have used this technique to selectively remove and isolate the capsular layer to analyze its components (Lemassu and Daffé, 1994; Lemassu et al., 1996; Ortalo-Magné et al., 1995). Others have reported that syringing through a 25-gauge needle produced no apparent disruption to the envelope on TEM (Stokes et al., 2004), but these samples were not evaluated further in terms of macrophage responses. We did not evaluate syringing, because it is not an approved method in our biosafety level 3 facility due to the risk of aerosolization and needle stick injuries. The physical forces used to disrupt clumps by this method might also result in envelope alterations, and investigators should consider this in their studies. Overall, we consider centrifuged samples as the least disrupted, but even centrifugation might disrupt the capsule, as do detergents that are commonly used in liquid cultures (and were used for all of our studies). Detergents are known to cause release of capsular components into the culture filtrate (Kalscheuer et al., 2019; Sani et al., 2010), although the impact on host interactions is relatively unexplored. The impact of detergent treatment on cytokine responses and vaccine responses have been evaluated, but after detergent treatment, single cell suspensions were generated by filtering, sonicating, or syringing (Prados-Rosales et al., 2016; Sani et al., 2010), complicating the interpretation.

Even if investigators had a non-disruptive way to isolate single cells, the behavior of single cells may not be the same as large aggregates that make up a substantial fraction of the unperturbed bacterial population. The aggregation state of Mtb has long been reported to be important to pathogenesis. For example, the observation that Mtb forms serpentine cords in vivo dates back to the earliest descriptions of the bacteria. Aggregated Mtb are found at the periphery of human necrotic granulomas, in alveolar macrophages of Mtb-infected patients, and are exhaled by infected individuals (Dinkele et al., 2021; Rodel et al., 2021; Ufimtseva et al., 2018). The literature describing the impact of aggregation on host interactions is difficult to interpret in light of our findings, as many of these studies used agitation with glass beads, filtration, sonication, or some combination of these procedures to generate the dispersed samples (Kolloli et al., 2021; Mahamed et al., 2017; Rodel et al., 2021). Thus, bacterial aggregation is likely an important virulence property of Mtb, which investigators overlook in the effort to generate single cell suspensions; at the same time, in generating single cell suspensions, investigators introduce the potential for experimental artifact.

It is possible that technical differences in how other laboratories sonicate or filter bacteria could result in findings that are different from ours. We used log phase cultures of H37Rv, HN878, and Erdman strains that had been grown with gentle agitation, a fatty acid source (oleic acid), and 0.05% Tyloxapol to infect mouse macrophages, but other investigators use different strains, frozen stocks, omit oleic acid, use different detergents, or infect human macrophages, all of which could lead to differences from our findings. An important conclusion of our findings is that investigators should fully report the methods that they use to grow and process mycobacteria and consider the impact of the methodology on their findings.

While sonication is an artificial stimulus, our findings suggest that Mtb keeps in check its massive pro-inflammatory potential by the organization and integrity of the envelope (Figure 7B). We imagine that by altering cell envelop architecture, Mtb tune their interactions to achieve the desired host response Garcia-Vilanova et al., 2019; for example, for initial infection and persistence, it may benefit the bacilli to minimize the TLR2-driven inflammatory response to promote immune evasion, whereas in order to drive tissue pathology and transmission, the bacilli may generate a hyperinflammatory phenotype (Chandra et al., 2022). While the Mtb cell wall is known to be dynamic (Dulberger et al., 2020), little is known about the structure and function of the cell wall during different in vivo contexts. To this end, a recent study evaluated the ultrastructure of the Mtb cell wall ex vivo from infected human sputum samples (Vijay et al., 2017). The characteristic three layers were found, and a reduction in the electron translucent layer was noted when bacilli were grown under stress conditions. Further in vivo studies investigating how Mtb regulates cell envelope architecture to modulate host inflammatory responses and deploy virulence lipids and protein effectors are needed.

RNA-seq data can be accessed in BioProject (Accession PRJNA851060; ID: 851060).

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Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further for publication by eLife.

Specifically, reviewers felt that your submission reports potentially important data however, the observations require further mechanistic insight to serve as a useful resource for the field. More detail is provided in the reviewer comments below.

Reviewer #1 (Recommendations for the authors):

The mycobacterial cell wall is complex, including an outer mycomembrane, non-covalently attached complex lipids, and depending upon growth conditions, a polysaccharide capsule. Multiple constituents of the cell wall have been shown to have impacts on host cells. In this manuscript, the authors investigate whether different methods used to prepare a single cell suspension of Mtb for infection studies change the mycobacterial cell wall in ways that lead to differential outcomes after infection. To do so, they compared sonication of bacteria followed by a low-speed spin with filtration through a 5uM filter. Sonicating followed by a low-speed spin resulted in an infection that was more inflammatory with higher induction of IL1, IL6, Nos2, and TNF. This increased inflammatory cytokine expression was TLR2 dependent. In addition, they show that sonicated bacteria grow in macrophages whereas filtered bacteria do not. However, the method of preparation of bacteria has no effect in on growth or inflammatory cytokine production in vivo in the mouse model. Finally, they show that mutants lacking the complex lipid PDIM are hyperinflammatory relative to wild type, as has been previously reported, but only when bacteria are prepared by sonication prior to infection. The major strength of this paper is the potential for providing clarity for the entire field around the impact of technical methods for preparing bacteria for infection during macrophage studies, which could bring more consistency and ability to interpret disparate results. The weaknesses are a lack of biological mechanistic insight, the lack of impact on in vivo infections and inconsistencies that make it difficult to draw clear conclusions from the data presented.

1. It is interesting that the authors present data showing a difference in growth in bone marrow derived macrophages in which sonicated or spun bacteria can grow, but filtered bacteria do no. Many labs do not sonicate bacterial stocks prior to macrophage infection, and even filter the cells, yet do see growth of bacteria in macrophages. In fact, from at least one previous publication from the author's lab, although the methods report that 5uM filtering was used to prepare the bacteria for macrophage infection, they observed growth of the bacteria in macrophages (PMID: 32636249). Can the authors explain this apparent discrepancy?

2. There are differences in the way bacteria that have been spun only (sp) behave compared with sonicated and spun (so/sp) vs filtered that make a clear understanding of the impact of these treatments on the bacteria difficult. For example, sp bacteria most closely resemble filtered when comparing inflammatory cytokine gene expression; however like so/sp bacteria they grow in macrophages. Sp bacteria also retain an outer layer (the composition of which is ambiguous) that so/sp bacteria do but do not have membrane blebs. Thus taken together, it is difficult to connect the observed differences in bacterial morphology with the different phenotypes seen during macrophage infection.

3. The authors show data that PDIM mutants are hyper-inflammatory relative to wild-type bacteria, but only if they have been sonicated. This is contradictory to a previous publication that showed that PDIM mutants elicit higher levels of TNFa (also examined in this study) during macrophage infections in which bacteria were not prepared by sonication (PMID: 34755600).

4. The authors present data showing that there is pro-inflammatory activity contained within the supernatants of bacteria that have been sonicated by applying supernatants from a bacterial suspension that has been passed through a 0.2uM filter to macrophages. However, it is not clear how much of the observed pro-inflammatory activity during infection itself comes from these soluble materials vs the bacteria themselves. If they wash the sonicated bacteria what do they see? It would be nice to see an experiment with so/sp bacteria, sups, and washed so/sp bacteria side by side.

Reviewer #2 (Recommendations for the authors):

The interaction of Mtb with macrophages is extensively studied by TB investigators and several studies have suggested that the methodology used to prepare Mtb for macrophage infections may influence the macrophage response. In this study the authors aimed to carefully examine this phenomenon with the lab-derived Mtb H37Rv strain. They found that disaggregation methods had a huge impact on macrophage responses in vitro and cautioned that this may lead to experimental artefacts. However, Mtb subjected to different disaggregation methods showed no difference in their in vivo growth and cytokine production in infected mice.

Strengths

1. The authors tackle an important problem that TB investigators face when performing in vitro macrophage infection studies that may lead to erroneous interpretation of data.

2. The data support the conclusions drawn.

3. TEM studies to examine damage to bacterial envelop by the two disintegration methods.

Weaknesses

1. The authors have not considered bacterial cell population heterogeneity.

2. Lack of a mechanism for the different in vitro and in vivo outcomes with sonicated and filtered Mtb.

3. No data with Mtb clinical strains.

In this study, the authors examined the impact of two routinely used bacterial disaggregation techniques on the subsequent interaction of Mtb with macrophages. They found that sonicated bacilli induced a strong hyperinflammatory response in BMDMs and still replicated normally in the macrophages. In stark contrast, Mtb that had been passed through a filter induced little inflammatory response in BMDMs and were also highly growth attenuated. Absence of PDIM impacted sonicated but not filtered bacteria. Interestingly, disaggregation methods did not impact growth in AMs or in vivo. In conclusion, the data presented here indicate that macrophage responses are strongly affected by the bacterial disaggregation methodology used and this may confound the results of Mtb-macrophage interaction studies.

Comments

1. The authors should rule out the possibility that their findings could be biased by cell population heterogeneity in Mtb H37Rv.

2. A lipid analysis of the bacteria after sonication and filtration would help substantiate the authors' conclusions.

3. Data from additional Mtb strains is required to strengthen the observations.

Reviewer #3 (Recommendations for the authors):

In this work, Mittal et al. determine how three commonly used methods for preparing Mtb for use in cellular and animal models of infection impact host response and infection outcomes. While largely technical, the key findings of the manuscript are important for understanding the existing literature of TB infection biology, and will undoubtedly inform how groups studying Mtb virulence in cellular and animal models perform and interpret their experiments in the future.

The primary comparisons were between sonication, filtration through a 5um filter, and centrifugation. Key findings:

1) Macrophages responded markedly differently to Mtb that were sonicated, filtered to make single-cell suspensions, or centrifuged- the primary initial outcome evaluated was comprehensive transcriptional profiling at 72 hours.

2) Mtb preparation using sonication results in enhanced inflammatory responses relative to uninfected or filtered cells. Outcomes evaluated include focused inflammatory gene transcriptional responses at 6 hours and 24 hours and TNF secretion as measured by FluoroDOT assays and TNF secretion and IL-1b secretion as measured by ELISA. Further, this enhanced inflammation is conferred by the sterile filtrate of the sonicated Mtb preparation, suggesting shedding of bacterial components into the media.

3) Mtb preparation using filtration results in reduced growth in bone marrow-derived macrophages but no impaired growth in alveolar macrophages or in vivo in mice.

4) TEM and uranyl acetate staining showed structural differences in the outer layers of Mtb prepared using sonication, filtration, and centrifugation. Sonicated Mtb had associated extra-bacterial vesicles; filtered Mtb had a thin outer coat suggesting loss of the outer membrane and capsule.

5) The impact of PDIM on early macrophage inflammatory responses as measured by targeted inflammatory gene expression and TNF FluorDOT assay was notable only for sonicated Mtb.

The finding that the preparation of Mtb changes inflammatory responses is compelling and well-supported by the transcriptional and cytokine data, in particular for the enhanced response to sonicated bacteria. The findings that this enhanced inflammation is partially TLR2 dependent and is associated with a soluble component of the bacterial preparation are similarly solid.

The finding that filtration results in reduced Mtb growth in BMDM relative to sonicated or centrifuged Mtb would be stronger if macrophage survival was assayed in parallel. At the MOIs used, Mtb infection is well-described to result in some degree of macrophage cell death, which can significantly impact recovery of Mtb from macrophage cell cultures. Clumping has also been described to have implications for macrophage cell death; given that the authors show different levels of clumping for bacteria prepared using each method, this is a particularly important outcome to measure in parallel with CFU.

The interpretation that the TEM findings and uranyl acetate staining (small extra bacterial vesicles for sonicated Mtb and loss of membrane/capsule for filtered Mtb) likely reflect the underlying reasons for differences in macrophage responses to the bacteria is consistent with the data, but not explicitly tested (and not readily testable).

The interpretation that differences in pre-existing macrophage inflammatory state in BMDM vs. alveolar macrophages drive the differences observed in the intracellular growth of filtered Mtb is also consistent with the data but not explicitly tested.

The overall conclusion that the specific methods used to prepare Mtb for infection impact macrophage responses and infection outcomes is solid and supported by the data. The conclusion that the method of bacterial preparation should be considered in both interpreting existing literature and experimental planning is also well-founded based on the data shown.

1) No testing of macrophage viability: Macrophage viability is another critical virulence related infection outcome, and can confound CFU measurements- in particular at higher MOIs and later timepoints. Measuring macrophage viability in parallel with CFU measurements would provide an important control for the CFU data and would offer insight into how the method of Mtb preparation impacts this additional infection outcome metric.

2) No testing of bacterial viability: While the relatively similar day 0 CFU in the macrophage infection assays suggest that similar numbers of live bacteria come out of each preparation method, the data would be stronger if bacterial viability were explicitly tested immediately after each preparation (using a live-dead reporter strain, or even CFU of the in vitro bacteria).

3) The hypothesis that the baseline inflammatory state of the macrophage influences whether filtered Mtb can grow is testable and potentially worth testing to more definitively support this idea. This could be done by pre-stimulating the macrophages with a range of that change the inflammatory and/or polarization state of the cells, then infecting with Mtb prepared using the three methods profiled.

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Essential revisions:

Reviewer #1 (Recommendations for the authors):

The mycobacterial cell wall is complex, including an outer mycomembrane, non-covalently attached complex lipids, and depending upon growth conditions, a polysaccharide capsule. Multiple constituents of the cell wall have been shown to have impacts on host cells. In this manuscript, the authors investigate whether different methods used to prepare a single cell suspension of Mtb for infection studies change the mycobacterial cell wall in ways that lead to differential outcomes after infection. To do so, they compared sonication of bacteria followed by a low-speed spin with filtration through a 5uM filter. Sonicating followed by a low-speed spin resulted in an infection that was more inflammatory with higher induction of IL1, IL6, Nos2, and TNF. This increased inflammatory cytokine expression was TLR2 dependent. In addition, they show that sonicated bacteria grow in macrophages whereas filtered bacteria do not. However, the method of preparation of bacteria has no effect in on growth or inflammatory cytokine production in vivo in the mouse model. Finally, they show that mutants lacking the complex lipid PDIM are hyperinflammatory relative to wild type, as has been previously reported, but only when bacteria are prepared by sonication prior to infection. The major strength of this paper is the potential for providing clarity for the entire field around the impact of technical methods for preparing bacteria for infection during macrophage studies, which could bring more consistency and ability to interpret disparate results. The weaknesses are a lack of biological mechanistic insight, the lack of impact on in vivo infections and inconsistencies that make it difficult to draw clear conclusions from the data presented.

We thank the reviewer for their thoughtful assessment. We agree that our findings may help explain inconsistencies in a large body of literature that has been published over decades studying the interaction between Mycobacterium tuberculosis (Mtb) and macrophages. Importantly, we hope our manuscript will ensure more robust and reproducible host-pathogen studies in the future. In our revised manuscript, we have tested two additional Mtb strains: HN878, a W-Beijing isolate that was isolated in a TB outbreak in Houston in the 1990s, and Erdman, a strain that is commonly used is laboratory studies. We found that the method of preparation has a similar impact on macrophage inflammatory responses and intracellular bacterial survival for these strains as for H37Rv (added as Figure 2 —figure supplement 1 and Figure 4EF). Thus, our findings are not specific to H37Rv and are likely to impact the field broadly.

In addition, we have revised the manuscript and added analyses to address all of the inconsistencies that were outlined below. We have included a figure that summarizes the data and more clearly integrates the impact of each preparation method into a coherent and mechanistic model (Figure 7). Our electron microscopy studies demonstrate that there is a substantial impact of these procedures on the structure of the mycobacterial cell envelope. The Mtb envelope has been shown by many other groups to contain a variety of pathogen associated molecular patterns (PAMPs) and virulence-associated proteins and lipids. We found that sonication alters the appearance of the envelope and increases macrophage inflammatory responses in a TLR2-dependent manner, consistent with the idea that it exposes these PAMPs. Similarly, we found that filtration removes components of the envelope and also attenuates intracellular survival of the bacilli. Our data support the idea that sonication and filtration are laboratory manipulations that, depending upon the question being investigated, generate experimental artifacts with the potential to lead to misinterpretation. We think that defining the lipid and/or protein changes induced by these laboratory manipulations is complex and beyond the scope of our studies, and, moreover, would not change the importance of our findings.

We initially included the in vivo studies, not because we think sonicated or filtered bacilli are biologically relevant for modeling natural infection, but we were concerned to what degree the artifacts related to preparation methods might also confound the in vivo literature. We found it reassuring that there did not appear to be a substantial impact on early in vivo infection. However, in thinking about the reviewers comments, we appreciate that we cannot exclude an impact of cell preparation on parameters we did not interrogate, such as inflammatory cell recruitment, the adaptive immune response, etc. In addition, we may have missed effects on cytokine responses, since we only measured a limited number of analytes, and we used a total lung sample, rather than interrogating directly infected cells (which would be technically difficult at low dose and early time points). Given these caveats, we have removed the in vivo data from the manuscript. Overall, we agree with the three reviewers that our macrophage findings are rigorous, well substantiated by the data, and have the potential for providing clarity to the field and for bringing more consistency in the future.

1. It is interesting that the authors present data showing a difference in growth in bone-marrow derived macrophages in which sonicated or spun bacteria can grow, but filtered bacteria do no. Many labs do not sonicate bacterial stocks prior to macrophage infection, and even filter the cells, yet do see growth of bacteria in macrophages. In fact, from at least one previous publication from the author's lab, although the methods report that 5uM filtering was used to prepare the bacteria for macrophage infection, they observed growth of the bacteria in macrophages (PMID: 32636249). Can the authors explain this apparent discrepancy?

We appreciate the reviewer’s careful attention to the data. We think that the text of the manuscript and the example that we showed overstated the impact of filtration on intracellular bacterial growth. Across all of the many experiments that we have done, the 55μm-filtered bacteria are impaired relative to non-filtered bacteria, but they do increase in bacterial burden a small amount over time. We have modified the text and replaced the initial figure with more representative data (Figure 4A). In addition, we have added data from HN878 and Erdman strains, which show a similar effect (Figure 4 E and F).

2. There are differences in the way bacteria that have been spun only (sp) behave compared with sonicated and spun (so/sp) vs filtered that make a clear understanding of the impact of these treatments on the bacteria difficult. For example, sp bacteria most closely resemble filtered when comparing inflammatory cytokine gene expression; however like so/sp bacteria they grow in macrophages. Sp bacteria also retain an outer layer (the composition of which is ambiguous) that so/sp bacteria do but do not have membrane blebs. Thus taken together, it is difficult to connect the observed differences in bacterial morphology with the different phenotypes seen during macrophage infection.

We appreciate that the multiple methods of preparation used in this study made it challenging for the reader to conceptualize the impact of each treatment. To address this, we now include a schematic diagram to show how sonicating, centrifuging, and filtering impact cell envelope appearance, macrophage gene expression, and intracellular growth within macrophages (Figure 7). We also now include in the studies bacteria that have been sonicated and filtered (5μmF) to show the impact of sonication (increased inflammatory gene expression) and filtering (reduced viability in macrophages) simultaneously (Figure 2A, C, D, Figure 3A-B, Figure 4A-C, E, F). We made substantial changes to the discussion of the manuscript to more clearly communicate one potential model that explains our data.

3. The authors show data that PDIM mutants are hyper-inflammatory relative to wild-type bacteria, but only if they have been sonicated. This is contradictory to a previous publication that showed that PDIM mutants elicit higher levels of TNFa (also examined in this study) during macrophage infections in which bacteria were not prepared by sonication (PMID: 34755600).

The referenced paper utilized PDIM-deficient ∆mas and ∆ppsD strains to study the TLR2-dependent host inflammatory response to Mtb. Our data are consistent with the data in that paper, as we have now clarified in the discussion. In that paper, the authors note, “Tnf did not cluster with 1B genes; instead Tnf was in cluster 3 (Figure 1A) together with genes minimally impacted by PDIM and ESX-1.” When they directly tested the impact of PDIM on Tnf expression by qPCR, there were no significant differences noted between H37Rv and the PDIM-deficient strains (see their Figure 3CDE). TNF secretion trended higher in the ∆ppsD relative to the WT strain, however this did not reach statistical significance (see their Figure 3G). In the discussion, the authors concluded that “PDIM and ESX-1 only minimally impact expression of the early TLR2-dependent gene cluster, suggesting that the inherent capacity of TLR2 to ‘recognize’ cognate ligand on the bacterium is similar between wild-type Mtb and ESX-1- or PDIM-mutant Mtb.” Overall, ours findings are consistent with theirs, even though there are a variety of differences between their experimental methods and ours (including how BMDMs were differentiated [M-CSF vs. L929 supernatant], time points examined, and MOI [5 vs. 10]).

4. The authors present data showing that there is pro-inflammatory activity contained within the supernatants of bacteria that have been sonicated by applying supernatants from a bacterial suspension that has been passed through a 0.2uM filter to macrophages. However, it is not clear how much of the observed pro-inflammatory activity during infection itself comes from these soluble materials vs the bacteria themselves. If they wash the sonicated bacteria what do they see? It would be nice to see an experiment with so/sp bacteria, sups, and washed so/sp bacteria side by side.

We appreciate the reviewer’s thoughts in this direction. Given that our electron microscopy data showed that the many of the membrane changes in the sonicated samples were associated with the bacterial surface, we did not think washing would effectively release substantial amounts for further study. In addition, if we washed the bacteria after preparing single cells, once we spun them down, they would again aggregate. However, to answer the reviewer’s concern, we have included the data from the so/sp bacteria and the supernatants side-by-side (now included in Figure 2E), which allows the reader to compare the pro-inflammatory activity from the bacilli versus extra-bacterial material. In addition, to better address this question, we exposed BMDMs to the sterile filtrate of so/sp bacteria, spin bacteria, or 5μmF bacteria and found that only the filtrate of the so/sp bacteria increased inflammatory gene expression (now included in Figure 2 —figure supplement 2). Given that the sterile filtrate of the spin bacteria did not induce inflammatory gene expression, we can more confidently attribute these changes to soluble factors following sonication.

Reviewer #2 (Recommendations for the authors):

The interaction of Mtb with macrophages is extensively studied by TB investigators and several studies have suggested that the methodology used to prepare Mtb for macrophage infections may influence the macrophage response. In this study the authors aimed to carefully examine this phenomenon with the lab-derived Mtb H37Rv strain. They found that disaggregation methods had a huge impact on macrophage responses in vitro and cautioned that this may lead to experimental artefacts. However, Mtb subjected to different disaggregation methods showed no difference in their in vivo growth and cytokine production in infected mice.

Strengths

1. The authors tackle an important problem that TB investigators face when performing in vitro macrophage infection studies that may lead to erroneous interpretation of data.

2. The data support the conclusions drawn.

3. TEM studies to examine damage to bacterial envelop by the two disintegration methods.

Weaknesses

1. The authors have not considered bacterial cell population heterogeneity.

2. Lack of a mechanism for the different in vitro and in vivo outcomes with sonicated and filtered Mtb.

3. No data with Mtb clinical strains.

We thank the reviewer for their thoughtful comments. We have now include data for the clinical W-Beijing isolate, HN878, which was recovered from a patient in Houston in the 1990s, as well as another commonly used laboratory strain, Erdman. We had similar findings with H37Rv, HNH878, and Erdman: sonicated bacilli were hyperinflammatory, while those that were filtered were attenuated in macrophages, indicating that our findings extend beyond H37Rv (added as Figure 2 —figure supplement 1 and Figure 4E and F).

In our new manuscript, we have considered bacterial heterogeneity, both incorporating the topic into the discussion and performing additional analysis (added as Figure 2C and Figure 5G). We agree that Mtb cultures are highly heterogenous and that bulk assays, such as intracellular growth, qPCR, and ELISA, fail to take this into account. We considered the possibility that a small minority of the so/sp bacilli are contributing to the heightened inflammatory response, but the Fluoro-dot data, which allows us to visualize at a single cell level, argue against this possibility. If there were a small population of bacilli in the filtered sample that were inhibiting the inflammatory response, then we would have expected that the samples that were mixed would behave like the filtered sample, but rather we saw an intermediate phenotype. Two aspects of heterogeneity that we assessed are clump size and capsule thickness, which are now quantified and included in Figure 2C and 5G, respectively. We found a significant reduction in the thickness of the capsule of the 5μmF bacteria compared to both so/sp and sp bacteria. For all preparation methods, however, there was a wide distribution in cell envelope thickness, which may contribute to heterogeneity in macrophage responses to bacilli on a cell-to-cell level. Finally, we also evaluated the number of bacteria per clump and did not find differences that could explain our findings, so heterogeneity in this aspect of the bacterial populations is unlikely to explain the differences in outcomes.

As we mentioned in the reply to Reviewer 1, we initially included the in vivo studies, not because we think sonicated or filtered bacilli are biologically relevant for modeling natural infection, but we were concerned that the artifacts created by preparation methods might also confound in vivo studies. We found it reassuring that there did not appear to be a substantial impact on early in vivo infection. There are many possible explanations for the in vivo and in vitro differences, including the difference between alveolar macrophages and bone marrow-derived macrophages, the impact of aerosolization, assessments over 7-14 days rather than 3-5 days, etc. However, in thinking about the reviewers’ comments, we appreciate that we cannot exclude an impact of cell preparation on parameters we did not interrogate, such as inflammatory cell recruitment, the adaptive immune response, etc. In addition, we may have missed effects on cytokine responses, since we only measured a limited number of analytes, and we used a total lung sample, rather than interrogating directly infected cells (which would be technically difficult at low dose and early time points). Given these caveats with the in vivo data, we have removed them from the manuscript.

In this study, the authors examined the impact of two routinely used bacterial disaggregation techniques on the subsequent interaction of Mtb with macrophages. They found that sonicated bacilli induced a strong hyperinflammatory response in BMDMs and still replicated normally in the macrophages. In stark contrast, Mtb that had been passed through a filter induced little inflammatory response in BMDMs and were also highly growth attenuated. Absence of PDIM impacted sonicated but not filtered bacteria. Interestingly, disaggregation methods did not impact growth in AMs or in vivo. In conclusion, the data presented here indicate that macrophage responses are strongly affected by the bacterial disaggregation methodology used and this may confound the results of Mtb-macrophage interaction studies.

Comments

1. The authors should rule out the possibility that their findings could be biased by cell population heterogeneity in Mtb H37Rv.

We have included discussion about the potential impact of heterogeneity, and to assess a potential impact of bacterial heterogeneity in our results, we further analyzed microscopy images to quantify variation in bacterial envelope thickness and used immunofluorescence to quantify clump size for each preparation method. When we evaluated capsule thickness, there was significant reduction in the thickness of the bacteria in the 5μmF samples compared to both so/sp and sp samples, but in all cases, there is a wide distribution, which may contribute to heterogeneity in macrophage responses on a cell-to-cell level. We considered the possibility that a small minority of the so/sp bacilli are contributing to the heightened inflammatory response, but the fluoro-dot data (which provided single cell resolution of TNF secretion) argue against this possibility. We also evaluated the number of bacteria per clump and filtered (5μmF and so/5μmF) preparations had slightly more single/doublets and slightly fewer clumps than the other samples (so/sp and sp; Figure 2C). But the aggregation status of different preparation did not explain the differences that we see in macrophage responses.

2. A lipid analysis of the bacteria after sonication and filtration would help substantiate the authors' conclusions.

Our electron microscopy studies demonstrate that there is an obvious structural impact of sonication and filtration on the mycobacterial cell envelope. We attempted to further characterize the differences in lipid composition, in collaboration with an expert in Mtb lipidomics (Fong-Fu Hsu, Washington University School of Medicine). It is standard in Mtb lipidomic studies to grow the bacilli on plates to eliminate need for detergents, which interfere with the mass spectrometry analysis. However, to compare the bacterial preparation methods described here requires adapting the analysis to liquid cultures. We were unable to establish conditions in which we could both reliably quantify and normalize across samples while also minimizing or removing the detergent so as to not interfere with the analysis. In the end, while we could see that 5umF bacilli lacked numerous envelope lipids, we were unable to obtain data that we consider publishable. We think that to rigorously define the lipid, protein and/or architectural changes induced by these laboratory manipulations is beyond the scope of these studies, and, importantly, would not change the importance and impact of our findings.

3. Data from additional Mtb strains is required to strengthen the observations.

To extend our findings to other laboratory strains and clinical strains, we measured intracellular growth of Mtb in BMDMs and BMDM gene expression using Erdman and HN878 strains alongside H37Rv and obtained comparable results. As in H37Rv, these strains elicited increased inflammatory gene expression in BMDMs when sonicated (Figure 2 —figure supplement 1). Further, the filtered bacteria were growth restricted in BMDMs (Figure 4E and F).

Reviewer #3 (Recommendations for the authors):

In this work, Mittal et al. determine how three commonly used methods for preparing Mtb for use in cellular and animal models of infection impact host response and infection outcomes. While largely technical, the key findings of the manuscript are important for understanding the existing literature of TB infection biology, and will undoubtedly inform how groups studying Mtb virulence in cellular and animal models perform and interpret their experiments in the future.

The primary comparisons were between sonication, filtration through a 5um filter, and centrifugation. Key findings:

1) Macrophages responded markedly differently to Mtb that were sonicated, filtered to make single-cell suspensions, or centrifuged- the primary initial outcome evaluated was comprehensive transcriptional profiling at 72 hours.

2) Mtb preparation using sonication results in enhanced inflammatory responses relative to uninfected or filtered cells. Outcomes evaluated include focused inflammatory gene transcriptional responses at 6 hours and 24 hours and TNF secretion as measured by FluoroDOT assays and TNF secretion and IL-1b secretion as measured by ELISA. Further, this enhanced inflammation is conferred by the sterile filtrate of the sonicated Mtb preparation, suggesting shedding of bacterial components into the media.

3) Mtb preparation using filtration results in reduced growth in bone marrow-derived macrophages but no impaired growth in alveolar macrophages or in vivo in mice.

4) TEM and uranyl acetate staining showed structural differences in the outer layers of Mtb prepared using sonication, filtration, and centrifugation. Sonicated Mtb had associated extra-bacterial vesicles; filtered Mtb had a thin outer coat suggesting loss of the outer membrane and capsule.

5) The impact of PDIM on early macrophage inflammatory responses as measured by targeted inflammatory gene expression and TNF FluorDOT assay was notable only for sonicated Mtb.

The finding that the preparation of Mtb changes inflammatory responses is compelling and well-supported by the transcriptional and cytokine data, in particular for the enhanced response to sonicated bacteria. The findings that this enhanced inflammation is partially TLR2 dependent and is associated with a soluble component of the bacterial preparation are similarly solid.

The finding that filtration results in reduced Mtb growth in BMDM relative to sonicated or centrifuged Mtb would be stronger if macrophage survival was assayed in parallel. At the MOIs used, Mtb infection is well-described to result in some degree of macrophage cell death, which can significantly impact recovery of Mtb from macrophage cell cultures. Clumping has also been described to have implications for macrophage cell death; given that the authors show different levels of clumping for bacteria prepared using each method, this is a particularly important outcome to measure in parallel with CFU.

The interpretation that the TEM findings and uranyl acetate staining (small extra bacterial vesicles for sonicated Mtb and loss of membrane/capsule for filtered Mtb) likely reflect the underlying reasons for differences in macrophage responses to the bacteria is consistent with the data, but not explicitly tested (and not readily testable).

The interpretation that differences in pre-existing macrophage inflammatory state in BMDM vs. alveolar macrophages drive the differences observed in the intracellular growth of filtered Mtb is also consistent with the data but not explicitly tested.

The overall conclusion that the specific methods used to prepare Mtb for infection impact macrophage responses and infection outcomes is solid and supported by the data. The conclusion that the method of bacterial preparation should be considered in both interpreting existing literature and experimental planning is also well-founded based on the data shown.

We thank the reviewer for the detailed summary of our findings. We agree that bacterial clumping likely has significant implications on macrophage-Mtb interactions, including cell death. To better evaluate this, we assessed macrophage cell death at days 0, 3, and 5 post-infection, alongside our intracellular bacterial survival assay. We did not find substantial differences based upon preparation method (now included as Figure 4C) that could explain our findings. We also utilized the microscopy images to evaluate heterogeneity in clump size between different preparations. The majority of the bacilli in all preparations were single or double, but there was considerable heterogeneity within each preparation, and there were more small clumps in the so/sp and sp samples. Since both so/sp and sp had similar distributions, clumpiness is unlikely to explain the macrophage inflammatory differences. We appreciate the reviewer’s comments regarding ex vivo infection of alveolar macrophages and in vivo infection of mice. On further review, our hypotheses were not well supported by the data. Along with the concerns of the other reviewers, we have removed the in vivo and alveolar macrophage data. We think that further phenotypic characterization of macrophage populations, while potentially interesting, would not change the conclusions of our manuscript or the impact of our study.

1) No testing of macrophage viability: Macrophage viability is another critical virulence related infection outcome, and can confound CFU measurements- in particular at higher MOIs and later timepoints. Measuring macrophage viability in parallel with CFU measurements would provide an important control for the CFU data and would offer insight into how the method of Mtb preparation impacts this additional infection outcome metric.

We used an ATP-based luminescence assay (CellTiterGlo, Promega) to evaluate viability of BMDMs infected at a multiplicity of infection (MOI) of 10 at 0, 3, and 5 days post-infection, alongside a replicate of our intracellular bacterial survival assay. There was a significant, but modest, increase in cell death in the spun bacilli at 5 days post-infection compared to uninfected macrophages and all other infected samples (perhaps related to a small number of large clumps in this preparation [see Figure 2C]). The cell death data has been added as Figure 4C. Overall, we conclude that the differences in intracellular growth were not explained by differences in macrophage viability (Figure 4C).

2) No testing of bacterial viability: While the relatively similar day 0 CFU in the macrophage infection assays suggest that similar numbers of live bacteria come out of each preparation method, the data would be stronger if bacterial viability were explicitly tested immediately after each preparation (using a live-dead reporter strain, or even CFU of the in vitro bacteria).

In addition to plating bacteria four hours after the macrophage infections, we verified that each preparation of Mtb used for the infection (input) produced roughly equivalent viable colonies when cultured on 7H10 plates. We found that the conversion between optical density and viable colony counts was slightly different for the filtered (5umF and so/5umF) bacilli compared to the other preparation (1 OD₆₀₀ = 3 x 10⁸ bacteria per mL for all preparation except filtered, which was 1 OD₆₀₀ = 2 x 10⁸ bacteria per mL). On the basis of viable colonies obtained on 7H10 plates, we calculated the MOI in each experiment. This has now been clarified in the methods. Furthermore, we freshly prepared so/sp, sp, or 5μmF and grew them in 7H9 liquid culture and confirmed that their growth patterns were indistinguishable from one another (Figure 4D).

3) The hypothesis that the baseline inflammatory state of the macrophage influences whether filtered Mtb can grow is testable and potentially worth testing to more definitively support this idea. This could be done by pre-stimulating the macrophages with a range of that change the inflammatory and/or polarization state of the cells, then infecting with Mtb prepared using the three methods profiled.

In our previous submission, we discussed differences between the response of BMDMs and alveolar macrophages. On further review, the conclusions that we drew were overly speculative. To address this, we have removed data related to ex vivo infection of alveolar macrophages, as well as the in vivo infections. While further testing of a variety of macrophages is possible, the preparation techniques we describe do not have clinical correlates to human TB disease, and we feel that this is beyond the scope of our paper. Recognizing that research groups use a variety of primary cells and often polarize cells before or after infection with Mtb, we recommend that they validate that their findings are not impacted by their method of bacterial preparation (or at least report their methodology and consider avoiding sonication and filtration).

Author details

1. Ekansh Mittal

   1. Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St Louis, United States
   2. Department of Molecular Microbiology, Washington University School of Medicine, St Louis, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing – review and editing

   Contributed equally with

   Andrew T Roth

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9034-033X

2. Andrew T Roth

   Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St Louis, United States

   Contribution

   Formal analysis, Investigation, Visualization, Writing - original draft, Writing – review and editing

   Contributed equally with

   Ekansh Mittal

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4239-7926

3. Anushree Seth

   Department of Mechanical Engineering and Materials Science, Institute of Materials Science and Engineering, Washington University in St. Louis, St Louis, United States

   Contribution

   Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   is currently employed with Auragent Bioscience LLC. The plasmonic-fluor technology used in the manuscript has been licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience LLC

4. Srikanth Singamaneni

   1. Department of Mechanical Engineering and Materials Science, Institute of Materials Science and Engineering, Washington University in St. Louis, St Louis, United States
   2. Siteman Cancer Center, Washington University, St. Louis, United States

   Contribution

   Resources, Supervision, Funding acquisition, Methodology, Writing – review and editing

   Competing interests

   is an inventor on a provisional patent related to plasmonic-fluor technology, and the technology has been licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience LLC. SS is a co-founder/shareholder of Auragent Bioscience LLC. SS along with Washington University may have financial gain through Auragent Bioscience LLC through this licensing agreement. These potential conflicts of interest have been disclosed and are being managed by Washington University in St. Louis

5. Wandy Beatty

   Department of Molecular Microbiology, Washington University School of Medicine, St Louis, United States

   Contribution

   Resources, Formal analysis, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Jennifer A Philips

   1. Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St Louis, United States
   2. Department of Molecular Microbiology, Washington University School of Medicine, St Louis, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing – review and editing

   For correspondence

   philips.j.a@wustl.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9476-0240

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