Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system

  1. Magda Luciana Atilano
  2. Pedro Matos Pereira
  3. Filipa Vaz
  4. Maria João Catalão
  5. Patricia Reed
  6. Inês Ramos Grilo
  7. Rita Gonçalves Sobral
  8. Petros Ligoxygakis
  9. Mariana Gomes Pinho
  10. Sérgio Raposo Filipe  Is a corresponding author
  1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Portugal
  2. University of Oxford, United Kingdom
  3. Centro de Recursos Microbiologicos (CREM), Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal
11 figures, 2 videos and 2 additional files

Figures

Staphylococcus aureus cells lacking the major autolysin Atl are better recognized by mCherry_PGRP-SA.

Exponentially growing bacteria from the parental NCTC8325-4 (NCTC), and its mutant strains lacking genes involved in cell wall metabolism (see main text for details) were incubated with …

https://doi.org/10.7554/eLife.02277.003
Better recognition of Staphylococcus aureus NCTCΔatl by PGRP-SA is not mediated by alterations in peptidoglycan muropeptide composition or lack of wall teichoic acids production.

(A) Staphylococcus aureus NCTCΔatl mutant has a similar peptidoglycan (PGN) muropeptide composition to the parental strain NCTC8325-4, as seen by HPLC analysis of mutanolysin-digested PGN. Roman …

https://doi.org/10.7554/eLife.02277.004
Amidase and glucosaminidase activities limit mCherry_PGRP-SA binding to the bacterial cell surface.

(A) Representation of the post-translational cleavage (black arrows) of the atl encoded protein. The processed amidase (AM, purple arrow) releases peptidoglycan (PGN) stem peptides by cutting the …

https://doi.org/10.7554/eLife.02277.005
Combination of purified staphylococcal AM and GL completely abolishes binding of mCherry_PGRP-SA to the surface Staphylococcus aureus atl null mutant cells.

(A) mCherry_PGRP-SA binding to NCTCΔatl bacteria pre-incubated with purified AM and GL (0.37 µM and 0.44 µM, respectively) showed that both enzymes can impair binding of mCherry_PGRP-SA to the cell …

https://doi.org/10.7554/eLife.02277.006
Activity, and not the presence of the Atl products, is required to avoid bacterial surface recognition by mCherry_PGRP-SA.

(A) NCTCΔatl cells were incubated with purified staphylococcal AM, GL, or PBS (negative control) and were either washed to clear these proteins from the cell surface, or unwashed to keep the …

https://doi.org/10.7554/eLife.02277.007
PGRP-SA is required to control infection by a Staphylococcus aureus mutant lacking the major autolysin Atl.

(A) Estimated survival curves of wild type (WT) 25174 flies infected with Atl producer NCTC8325-4 and NCTCΔatl strains were statistically different (p<0.005) and are indicated by asterisks. Absence …

https://doi.org/10.7554/eLife.02277.008
Lack of either Atl amidase or glucosaminidase activity, which leads to different types of released peptidoglycan fragments, results in decreased virulence of Staphylococcus aureus.

(A) Estimated survival curves of wild type (WT) 25174 flies infected with atl mutant strains impaired in amidase activity (AMH265A), glucosaminidase activity (GLE1128A), or both (AMH265AGLE1128A), …

https://doi.org/10.7554/eLife.02277.009
Atl products secreted by Staphylococcus aureus cells protect atl null mutant cells from PGRP-SA recognition and allow S. aureus to establish a successful infection in Drosophila.

(A) NCTCΔatl Staphylococcus aureus cells were incubated with TSB medium (control) or with supernatants (sterilized by filtration) from cultures of NCTC8325-4 (NCTCsup, containing atl encoded …

https://doi.org/10.7554/eLife.02277.010
Time-lapse microscopy showing that atl encoded proteins can mediate release of mCherry_PGRP-SA previously bound to the surface of atl null mutant Staphylococcus aureus cells.

NCTCΔatl cells, labeled with mCherry-PGRP-SA, were placed on top of a thin layer of agarose containing filter-sterilized supernatant of cultures of NCTCΔatl mutant (A, Video 1) or NCTC8325-4 …

https://doi.org/10.7554/eLife.02277.011
Atl encoded proteins are required to conceal CA-MRSA virulent strains from the Drosophila immune system.

(A) Staphylococcus aureus CA-MRSA MW2 strain and its atl null mutant, MW2Δatl, were incubated with mCherry_PGRP-SA in 96-well plates. The average amount of mCherry_PGRP-SA bound to bacteria in each …

https://doi.org/10.7554/eLife.02277.014
LytA activity can prevent Streptococcus pneumoniae and Staphylococcus aureus recognition by mCherry_PGRP-SA.

(A) Streptococcus pneumoniae parental strain R36A, a lytA null mutant (R36AΔlytA), and a complemented strain, expressing LytA from a replicative plasmid (R36AΔlytAplytA), were incubated with …

https://doi.org/10.7554/eLife.02277.015

Videos

Video 1
https://doi.org/10.7554/eLife.02277.012
Video 2
https://doi.org/10.7554/eLife.02277.013

Additional files

Supplementary file 1

Strains and plasmids used in this study.

https://doi.org/10.7554/eLife.02277.016
Supplementary file 2

Primers used in this study.

https://doi.org/10.7554/eLife.02277.017

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