Toxin Mediates Sepsis Caused by Methicillin-Resistant Staphylococcus epidermidis.

Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the psm-mec locus that have been reported in S. aureus strains, inasmuch as our findings suggest that the psm-mec locus may exert effects in the background of S. aureus strains that differ from its original role in the CNS environment due to originally "unintended" interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.

Antimicrobial Peptide Resistance Mechanism Contributes to Staphylococcus aureus Infection.

Antimicrobial peptides (AMPs) constitute an important part of innate host defense. Possibly limiting the therapeutic potential of AMPs is the fact that bacteria have developed AMP resistance mechanisms during their co-evolution with humans. However, there is no direct evidence that AMP resistance per se is important during an infection. Here we show that the Staphylococcus aureus Pmt ABC transporter defends the bacteria from killing by important human AMPs and elimination by human neutrophils. By showing that Pmt contributes to virulence during skin infection in an AMP-dependent manner, we provide evidence that AMP resistance plays a key role in bacterial infection.

Integration of cell wall synthesis and chromosome segregation during cell division in Caulobacter.

To divide, bacteria must synthesize their peptidoglycan (PG) cell wall, a protective meshwork that maintains cell shape. FtsZ, a tubulin homolog, dynamically assembles into a midcell band, recruiting division proteins, including the PG synthases FtsW and FtsI. FtsWI are activated to synthesize PG and drive constriction at the appropriate time and place. However, their activation pathway remains unresolved. In Caulobacter crescentus, FtsWI activity requires FzlA, an essential FtsZ-binding protein. Through time-lapse imaging and single-molecule tracking of Caulobacter FtsW and FzlA, we demonstrate that FzlA is a limiting constriction activation factor that signals to promote conversion of inactive FtsW to an active, slow-moving state. We find that FzlA interacts with the DNA translocase FtsK and place FtsK genetically in a pathway with FzlA and FtsWI. Misregulation of the FzlA-FtsK-FtsWI pathway leads to heightened DNA damage and cell death. We propose that FzlA integrates the FtsZ ring, chromosome segregation, and PG synthesis to ensure robust and timely constriction during Caulobacter division.

E. coli FtsN coordinates synthesis and degradation of septal peptidoglycan by partitioning between a synthesis track and a denuded glycan track.

The E. coli cell division protein FtsN was proposed to coordinate septal peptidoglycan (sPG) synthesis and degradation to ensure robust cell wall constriction without lethal lesions. Although the precise mechanism remains unclear, previous work highlights the importance of two FtsN domains: the E domain, which interacts with and activates the sPG synthesis complex FtsWIQLB, and the SPOR domain, which binds to denuded glycan (dnG) strands, key intermediates in sPG degradation. Here, we used single-molecule tracking of FtsN and FtsW (a proxy for the sPG synthesis complex FtsWIQLB) to investigate how FtsN coordinates the two opposing processes. We observed dynamic behaviors indicating that FtsN's SPOR domain binds to dnGs cooperatively, which both sequesters the sPG synthesis complex on dnG (termed as the dnG-track) and protects dnGs from degradation by lytic transglycosylases (LTs). The release of the SPOR domain from dnGs leads to activating the sPG synthesis complex on the sPG-track and simultaneously exposing those same dnGs to degradation. Furthermore, FtsN's SPOR domain self-interacts and facilitates the formation of a multimeric sPG synthesis complex on both tracks. The cooperative self-interaction of the SPOR domain creates a sensitive switch to regulate the partitioning of FtsN between the dnG- and sPG-tracks, thereby controlling the balance between sequestered and active populations of the sPG synthesis complex. As such, FtsN coordinates sPG synthesis and degradation in space and time.

The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria.

Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37°C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence.

Conformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism.

The bacterial divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during division. Here, we present a model of the structure and dynamics of the core complex of the E. coli divisome, supported by a combination of structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis. We focus on the septal cell wall synthase complex formed by FtsW and FtsI, and its regulators FtsQ, FtsL, FtsB, and FtsN. The results indicate extensive interactions in four regions in the periplasmic domains of the complex. FtsQ, FtsL, and FtsB support FtsI in an extended conformation, with the FtsI transpeptidase domain lifted away from the membrane through interactions among the C-terminal domains. FtsN binds between FtsI and FtsL in a region rich in residues with superfission (activating) and dominant negative (inhibitory) mutations. Mutagenesis experiments and simulations suggest that the essential domain of FtsN links FtsI and FtsL together, potentially modulating interactions between the anchor-loop of FtsI and the putative catalytic cavity of FtsW, thus suggesting a mechanism of how FtsN activates the cell wall synthesis activities of FtsW and FtsI.

FtsN maintains active septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthases in E. coli.

FtsN plays an essential role in promoting the inward synthesis of septal peptidoglycan (sPG) by the FtsWI complex during bacterial cell division. How it achieves this role is unclear. Here we use single-molecule tracking to investigate FtsN's dynamics during sPG synthesis in E. coli. We show that septal FtsN molecules move processively at ~9 nm s-1, the same as FtsWI molecules engaged in sPG synthesis (termed sPG-track), but much slower than the ~30 nm s-1 speed of inactive FtsWI molecules coupled to FtsZ's treadmilling dynamics (termed FtsZ-track). Importantly, processive movement of FtsN is exclusively coupled to sPG synthesis and is required to maintain active sPG synthesis by FtsWI. Our findings indicate that FtsN is part of the FtsWI sPG synthesis complex, and that while FtsN is often described as a "trigger" for the initiation for cell wall constriction, it must remain part of the processive FtsWI complex to maintain sPG synthesis activity.

Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW.

Synthesis of septal peptidoglycan (sPG) is crucial for bacterial cell division. FtsW, an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a peptidoglycan glycosyltransferase (PGTase). Despite its importance, the septal PGTase activity of FtsW has not been demonstrated in vivo. How its activity is spatiotemporally regulated in vivo has also remained elusive. Here, we confirmed FtsW as an essential septum-specific PGTase in vivo using an N-acetylmuramic acid analogue incorporation assay. Next, using single-molecule tracking coupled with genetic manipulations, we identified two populations of processively moving FtsW molecules: a fast-moving population correlated with the treadmilling dynamics of the essential cytoskeletal FtsZ protein and a slow-moving population dependent on active sPG synthesis. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving population. Our results suggest a two-track model, in which inactive sPG synthases follow the 'Z-track' to be distributed along the septum and FtsN promotes their release from the Z-track to become active in sPG synthesis on the slow 'sPG-track'. This model provides a mechanistic framework for the spatiotemporal coordination of sPG synthesis in bacterial cell division.

Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism.

The FtsZ protein is a central component of the bacterial cell division machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ's treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme's diffusion and FtsZ's treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.

PSM-Mec-A Virulence Determinant that Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci.

PSM-mec is a secreted virulence factor that belongs to the phenol-soluble modulin (PSM) family of amphipathic, alpha-helical peptide toxins produced by Staphylococcus species. All known PSMs are core genome-encoded with the exception of PSM-mec, whose gene is found in specific sub-types of SCCmec methicillin resistance mobile genetic elements present in methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci. In addition to the cytolytic translational product, PSM-mec, the psm-mec locus encodes a regulatory RNA. In S. aureus, the psm-mec locus influences cytolytic capacity, methicillin resistance, biofilm formation, cell spreading, and the expression of other virulence factors, such as other PSMs, which results in a significant impact on immune evasion and disease. However, these effects are highly strain-dependent, which is possibly due to differences in PSM-mec peptide vs. psm-mec RNA-controlled effects. Here, we summarize the functional properties of PSM-mec and the psm-mec RNA molecule and their roles in staphylococcal pathogenesis and physiology.