A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx

Suggestions for improvement:

– The authors need to run time-kill assays where in the interaction of popuCATH with antimicrobial is tested. This will provide some evidence if there is a future for this peptide in therapeutics.

We tested the interaction of PopuCATH (200 µg/mL) with ampicillin (0.5×MIC, 1×MIC, 2×MIC) against E. coli ATCC25922 by time-kill assay. We found that the presence of PopuCATH (200 µg/mL) did not promote the clearance of E. coli by 0.5×MIC, 1×MIC, 2×MIC of ampicillin, respectively. Therefore, it is not a good evidence for usage of PopuCATH and antimicrobial in combination, and we did not show this data as well as discuss this data in the revised manuscript.

– The authors cannot discuss on the anti-inflammatory activity of popuCATH since the attenuation of the inflammation may be secondary to the decrease of bacterial growth.

We have revised it in the manuscript as follows:

“In mouse model, the production of chemokines/cytokines in mouse abdominal cavity peaked at 4 h post injection of PopuCATH. The dynamic of CXCL1, CXCL2, and CXCL3 production is consistent with the dynamic of neutrophil, monocyte/macrophage recruitment in the mouse abdominal cavity and peripheral blood. Although the pretreatment with PopuCATH significantly induced the production of chemokines (CXCL1, CXCL2, and CXCL3) as well as pro-inflammatory cytokines (IL-1β and IL-6), PopuCATH ultimately attenuated the inflammatory response by decrease of TNF-α, IL-1β and IL-6 levels post Gram-negative and Gram-positive bacterial infection. Cathelicidins are able to block Toll like receptor (TLR)-mediated inflammatory responses, including those mediated by TLR2 and TLR4 (Coorens et al., 2017; Mookherjee et al., 2006; Wei et al., 2013). In this study, PopuCATH didn’t affect LTA- and LPS-stimulated inflammatory responses in mouse peritoneal macrophages (Figure 8—figure supplement 3), suggesting that the anti-inflammatory effects of PopuCATH are independent of TLRs, and the attenuation of the inflammation may be secondary to the decrease of bacterial growth.”

– It is very difficult to explain the total absence of effect when popuCATH is given after induction of infection or in parallel to bacterial challenge. This diversity generates ambiguity on the real physiological meaning of this peptide.

At the dose of 10 mg/kg, PopuCATH did not exhibit therapeutic efficacy against bacterial infection. In order to evaluate whether it has therapeutic efficacy at high doses, we increased the dose of PopuCATH up to 20 and 40 mg/kg. We found that 20 mg/kg and 40 mg/kg of PopuCATH significantly reduced the bacterial load when it was given at 4 h after E. coli inoculation (Figure 4—figure supplement 2). It is possibly that bacteria have colonized in mice at 4 h after bacterial inoculation, which need a higher dose of PopuCATH to drive more phagocytes for bacterial clearance. And we have added this finding in the Discussion section.

– Does popuCHATH has more than one gene per chromosome like defensins?

As described in the second paragraph of the Introduction section, we have identified a cathelicidin antimicrobial peptide from the skin of Polypedates puerensis previously (PMID: 28593346), implying that PopuCHATH probably has more than one gene per chromosome.

– In this study, a total of 16 amino acids at the N-terminus of the purified peptide were determined as SRGGRGGRGGGGSRGG by automated Edman degradation. The authors should have designed an antisense primer according to this partial mature peptide. However, the authors designed an antisense primer (5'-TTGTCTGCCTCCTCGGCTTCC-3') according to the conserved domain of amphibian cathelicidins. Why?

We have designed series of degenerate primers based on the amino acid sequence at the N-terminus. But we failed to clone the cDNA sequence. It is possibly that the amino acid sequence at the N-terminus of PopuCHATH is a glycine-rich peptide fragment, and the degenerate primers based on this fragment have high degeneracy. Therefore, we then designed an antisense primer (5'-TTGTCTGCCTCCTCGGCTTCC-3') according to the conserved domain of amphibian cathelicidins.

– In Figures 2A, 2B, 2C, 3A, 3C, 3D, 3E, and Supplementary file 3, the highest concentration of PopuCATH is 200 µg/mL. Why did the authors select 200 µg/mL, other than 300, 400, 500…µg/mL as the highest concentration?

According to a previous study (PMID: 17384586), we selected 200 µg/mL as the highest concentration for antimicrobial assay in vitro. Accordingly, if a peptide (drug) did not exhibit antimicrobial activity at a concentration up to 200 µg/mL in vitro, we usually think that it lacks direct antimicrobial activity. Based on the highest concentration used in antimicrobial assay in vitro, we also selected 200 µg/mL as the highest concentration for side effects assay in vitro.

– In both frogs and mice, PopuCATH (10 mg/kg) treatment at 8 h or 4 h before bacterial inoculation significantly reduced the bacterial load in tree frogs, but PopuCATH treatment at 4 h after bacterial inoculation did not significantly reduce the bacterial load. The results indicated that PopuCATH exhibited prophylactic efficacy, but not therapeutic efficacy, against bacterial infection. I would like to known the possible reasons why PopuCATH has prophylactic efficacy, but not therapeutic efficacy. At least, the authors should discuss the data.

In the last version of our manuscript, PopuCATH did not exhibit therapeutic efficacy against bacterial infection at dose of 10 mg/kg. In order to evaluate whether it has therapeutic efficacy at high doses, we increased the dose of PopuCATH in the revised manuscript. When the dose of PopuCATH was increased to 20 mg/kg, PopuCATH significantly reduced the bacterial load when it was given at 4 h after bacterial inoculation (Figure 4—figure supplement 2).

– In Figure 8, PopuCATH-mediated chemokine/cytokine production in macrophages and mice is somewhat different. What is(are) the possible reason(s)? I suggest the authors discuss this difference.

The expression profiles of chemokines/cytokines in vitro and in vivo are somewhat different, but we observed that the profiles of the major chemokines/cytokines induced by PopuCATH, such as CXCL1, CXCL2, and CXCL3, are similar. It is possibly that PopuCATH-induced chemokines/cytokines in vivo are consumed timely. In addition, macrophage is the unique effector cell type of PopuCATH in vitro. While there are many other cell types in vivo, such as monocytes and neutrophils, and we cannot exclude these cells are responsive to PopuCATH and subsequently produce chemokines/cytokines. These points may explain the subtle differences of PopuCATH-mediated chemokine/cytokine production in macrophages and mice. And we have discussed it in the Discussion section.

– A previous study showed that a frog-derived glycine-rich cathelicidin exhibited direct antimicrobial activity (Hao et al., 2012). In this study, PopuCATH is also a glycine-rich cathelicidin. But PopuCATH did not show any direct antimicrobial activity at concentration up to 200 µg/mL. Why? Please discuss this phenomenon.

We have discussed it in the Discussion section as follows:

“The first frog-derived cathelicidin is also rich in glycine residues. But it has different amino acid sequence with PopuCATH and exhibits direct anti-bacterial activity unlike PopuCATH (Hao et al., 2012). Cathelicidin antimicrobial peptides display a high structural diversity, and the diverse structures are responsible for their diverse functions. Accordingly, it is not difficult to understand that these two frog cathelicidins have different functions against bacteria.”

– Line 103-104, the authors described that a novel naturally occurring cathelicidin was identified from the skin of tree frog, P. puerensis. Line 497, the authors described that synthetic peptides were purchased from Synpeptide Co. Ltd. That is to say, some results were obtained by using naturally occurring PopuCATH, and the others were obtained by using synthetic PopuCATH. Please describe which results were obtained by using naturally occurring/synthetic PopuCATH in the responding Materials and methods section.

Naturally occurring PopuCATH was just used in mass spectrometry and Edman degradation assay. Synthetic PopuCATH was used in other experiments. And we have described this point in the Materials and methods section.

– In Figure 8, the mRNA levels of chemokines/cytokines in macrophages induced by PopuCATH were tested by Q-PCR. Please list the primers for Q-PCR assay.

We are sorry to lose the primers for Q-PCR assay, and we have listed the primers in Supplementary file 5.

– To assay the effector cells, the protective efficacy of PopuCATH was evaluated in neutrophil or monocyte/macrophage depletion mice by intraperitoneal injection of anti-Ly6G antibody (clone 1A8, BioXcell) and anti-CSF1R antibody (clone AFS98, BioXCell), respectively. However, I the scavenging efficiency of intraperitoneal injection of anti-Ly6G antibody (clone 1A8, BioXcell) and anti-CSF1R antibody (clone AFS98, BioXCell) should be provided, respectively.

We have provided the scavenging efficiency of intraperitoneal injection of anti-Ly6G antibody (clone 1A8, BioXcell) and anti-CSF1R antibody (clone AFS98, BioXCell) in the revised manuscript as shown in Figure 6—figure supplement 1.