Cell-wall peptidoglycan (PG) of Gram-positive bacteria is a strong and elastic multi-layer designed to resist turgor pressure and determine the cell shape and growth. Despite its crucial role, its architecture remains largely unknown. Here using high-resolution multiparametric atomic force microscopy (AFM), we studied how the structure and elasticity of PG change when subjected to increasing turgor pressure in live Group B Streptococcus. We show a new net-like arrangement of PG, which stretches and stiffens following osmotic challenge. The same structure also exists in isogenic mutants lacking surface appendages. Cell aging does not alter the elasticity of the cell wall, yet destroys the net architecture and exposes single segmented strands with the same circumferential orientation as predicted for intact glycans. Together, we show a new functional PG architecture in live Gram-positive bacteria.
Background Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone.Methodology/Principal FindingsIn this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity.Conclusions/SignificanceThese results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context.
The outcome of an antibiotic treatment on the growth capacity of bacteria is largely dependent on the initial population size (Inoculum Effect). We characterized and built a model of this effect in E. coli cultures using a large variety of antimicrobials, including conventional antibiotics, and for the first time, cationic antimicrobial peptides (CAMPs). Our results show that all classes of antimicrobial drugs induce an inoculum effect, which, as we explain, implies that the dynamic is bistable: For a range of anti-microbial densities, a very small inoculum decays whereas a larger inoculum grows, and the threshold inoculum depends on the drug concentration. We characterized three distinct classes of drug-induced bistable growth dynamics and demonstrate that in rich medium, CAMPs correspond to the simplest class, bacteriostatic antibiotics to the second class, and all other traditional antibiotics to the third, more complex class. These findings provide a unifying universal framework for describing the dynamics of the inoculum effect induced by antimicrobials with inherently different killing mechanisms.
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