Biology subjects

Foster, S. J.

Publications and source records attributed to Foster, S. J..

7 recordsLinked to original sources

bioRxiv2026

Morphological transformation in Helicobacter pylori is a dynamic process leading to two types of coccoid

The helical shape of Helicobacter pylori is crucial for successful colonization of the human stomach. However, this pathogen can shapeshift into another form termed the "coccoid form" with a spherical shape, through a mechanism that remains elusive. Here, by a combination of fluorescence microscopy using fluorescent D-aminoacids, cryoelectron and atom force microscopy, we explored the dynamics of coccoid formation in H. pylori through interrogation of the peptidoglycan layer. Contrary to the widely held hypothesis, we showed that helical-rod H. pylori transformed into a coccoid without transiting through a U-form. We show that U-forms, characterized by a U-shaped peptidoglycan with enlarged periplasmic space, altered genetic material, and red autofluorescence, are the output of a parallel pathway, which, unlike the coccoid pathway, is independent of the HdpA/Csd3 peptidoglycan endopeptidase. Coccoid formation occurred along a rigid timeline, by bulging of the cytoplasmic membrane through a peptidoglycan crack, resulting in a spheroplast-like structure with the peptidoglycan stacked into a thick layer near the original cell poles. Resistance of that structure against lysis likely involves a switch in metabolic profile reminiscent of bacteria in dormancy, with a notable accumulation of lysophospholipids, demonstrated in this work. Altogether, the ultrastructure and properties of H. pylori coccoids evidenced here are compatible with a role of this form in relapse after antibiotic treatment. SIGNIFICANCE STATEMENTUpon prolonged growth, stomach pathogen Helicobacter pylori undergoes a morphological change from a helical to a spherical form called coccoid, which may be involved in bacterial persistence and immune evasion. The pathway leading to this form, as well as its precise architecture, were unclear. In this work, we show by different microscopy techniques that the transition to coccoid does not involve a U-shaped intermediate as proposed before, but is triggered by progressive thinning, due to the activity of endopeptidase HdpA/Csd3, of the peptidoglycan meshwork that normally protects the cell, eventually leading to rupture. Because of the hole thus created, peptidoglycan can no longer contain the osmotic pressure in the cytoplasm, which leaks out within a membrane bulge, to eventually give rise to a kind of sphaeroplast, expected to be insensitive to cell wall-targeted antibiotics.

microbiology↗

bioRxiv2023

The ClpX chaperone and a hypermorphic FtsA variant with impaired self-interaction are mutually compensatory for coordinating Staphylococcus aureus cell division

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome, however, the exact role of molecular chaperones in this critical process remains unclear. In the important pathogenic bacterium Staphylococcus aureus, the ClpX chaperone is essential for growth at 30{degrees}C and microscopic analyses suggested that ClpX plays a temperature-dependent role in cell division. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of cell division by showing that a spontaneous G325V substitution in the ATP-binding domain of the essential FtsA cell division protein rescues growth and septum synthesis in a Staphylococcus aureus clpX mutant. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with E. coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division. IMPORTANCEEssential biological processes, such as cell division, are performed by multiple proteins working together in dynamic functional complexes. In eukaryotic cells, the disassembly of such molecular machines is often assisted by molecular chaperones capable of unfolding proteins. The ClpX unfoldase is conserved from bacteria to humans, however, the roles of ClpX in bacterial cell biology remain relatively unexplored. By combining genetic methods with super-resolution microscopy techniques, we show here that ClpX and a mutant variant of the essential cell division protein FtsA mutually compensate for each other in controlling cell division of the pathogenic bacterium Staphylococcus aureus. The selected FtsAG325V variant has diminished self-interactions and restored the aberrant FtsA dynamics in clpX cells suggesting that ClpX promotes cell division by antagonizing FtsA protein interactions. This study, for the first time, illuminates the important role of protein unfoldases in the functioning of the divisome, a multienzyme complex fundamental to bacterial reproduction.

molecular biology↗

bioRxiv2023

The roles of GpsB and DivIVA in Staphylococcus aureus growth and division

The spheroid bacterium S. aureus is often used as a model of morphogenesis due to its apparent simple cell cycle. S. aureus has many cell division proteins that are conserved across bacteria alluding to common functions. However, despite intensive study we still do not know the roles of many of these components. Here we have examined the functions of the paralogues DivIVA and GpsB in the S. aureus cell cycle. Cells lacking gpsB display a more spherical phenotype than wild type, associated with a decrease in peripheral cell wall peptidoglycan synthesis. This correlates with an increased localisation of penicillin binding proteins at the developing septum, notably PBPs 2 and 3. Our results highlight the role of GpsB as an apparent regulator of cell morphogenesis in S. aureus.

microbiology↗

bioRxiv2021

PBP1 of Staphylococcus aureus has multiple essential functions in cell division

Bacterial cell division is a complex process requiring the coordination of multiple components, to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multi- stage PG architecture that develops during septation. Penicillin binding proteins (PBPs) are essential for the final steps of PG biosynthesis - their transpeptidase activity links together the peptide sidechain of nascent glycan strands together. PBP1 is required for cell division in S. aureus and here we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thus coordinate the cell division process. The transpeptidase activity of PBP1 is also essential but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific {beta}-lactam, meropenem. Together these results lead to a model for septal PG synthesis where PBP1 enzyme activity is responsible for the characteristic architecture of the septum and PBP1 protein molecules coordinate cell division allowing septal plate formation.

microbiology↗

bioRxiv2020

Humanisation of the zebrafish C5a receptor confers targeting by human-specific staphylococcal virulence factors.

Staphylococcus aureus infects approximately 30% of the human population and causes a spectrum of pathologies ranging from mild skin infections to life-threatening invasive diseases. The strict host specificity of its virulence factors has severely limited the accuracy of in vivo models for the development of vaccines and therapeutics. To resolve this, we generated a humanised zebrafish model and determined that neutrophil-specific expression of the human C5a receptor conferred susceptibility to the S. aureus toxins PVL and HlgCB, leading to reduced neutrophil numbers at the site of infection and increased infection-associated mortality as a direct result of the interaction between S. aureus and the receptor. These results show that humanised zebrafish provide a valuable platform to study the contribution of human-specific S. aureus virulence factors to infection in vivo that could facilitate the development of novel therapeutic approaches and essential vaccines.

immunology↗

bioRxiv2018

A transgenic zebrafish line for in vivo visualisation of neutrophil myeloperoxidase

The neutrophil enzyme myeloperoxidase (MPO) is a major enzyme made by neutrophils to generate antimicrobial and immunomodulatory compounds, notably hypochlorous acid (HOCl), amplifying their capacity for destroying pathogens and regulating inflammation. Despite its roles in innate immunity, the importance of MPO in preventing infection is unclear, as individuals with MPO deficiency are asymptomatic with the exception of an increased risk of candidiasis. Dysregulation of MPO activity is also linked with inflammatory conditions such as atherosclerosis, emphasising a need to understand the roles of the enzyme in greater detail. Consequently, new tools for investigating granular dynamics in vivo can provide useful insights into how MPO localises within neutrophils, aiding understanding of its role in preventing and exacerbating disease. The zebrafish is a powerful model for investigating the immune system in vivo, as it is genetically tractable, and optically transparent. To visualise MPO activity within zebrafish neutrophils, we created a genetic construct that expresses human MPO as a fusion protein with a C-terminal fluorescent tag, driven by the neutrophil-specific promoter lyz. After introducing the construct into the zebrafish genome by Tol2 transgenesis, we established the Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496 line, and confirmed transgene expression in zebrafish neutrophils. We observed localisation of MPO-mEmerald within a subcellular location resembling neutrophil granules, mirroring MPO in human neutrophils. In Spotless (mpxNL144) larvae - which express a non-functional zebrafish myeloperoxidase - the MPO-mEmerald transgene does not disrupt neutrophil migration to sites of infection or inflammation, suggesting that it is a suitable line for the study of neutrophil granule function. We present a new transgenic line that can be used to investigate neutrophil granule dynamics in vivo without disrupting neutrophil behaviour, with potential applications in studying processing and maturation of MPO during development.

immunology↗

bioRxiv2018

SosA inhibits cell division in Staphylococcus aureus in response to DNA damage

Inhibition of cell division is critical for cell viability under DNA damaging conditions. In bacterial cells, DNA damage induces the SOS response, a process that inhibits cell division while repairs are being made. In coccoid bacteria, such as the human pathogen Staphylococcus aureus, the process remains poorly understood. Here we have characterized an SOS-induced cell-division inhibitor, SosA, in S. aureus. We find that in contrast to the wildtype, sosA mutant cells continue division under DNA damaging conditions with decreased viability as a consequence. Conversely, overproduction of SosA leads to cell division inhibition and reduced growth. The SosA protein is localized in the bacterial membrane and mutation of an extracellular amino acid, conserved between homologs of other staphylococcal species, abolished the inhibitory activity as did truncation of the C-terminal 30 amino acids. In contrast, C-terminal truncation of 10 amino acids lead to SosA accumulation and a strong cell division inhibitory activity. A similar phenotype was observed upon expression of wildtype SosA in a mutant lacking the membrane protease, CtpA. Thus, the extracellular C-terminus of SosA is required both for cell-division inhibition and for turnover of the protein. Functional studies showed that SosA is likely to interact with one or more divisome components and, without interfering with early cell-division events, halts cell division at a point where septum formation is initiated yet being unable to progress to septum closure. Our findings provide important insights into cell-division regulation in staphylococci that may foster development of new classes of antibiotics targeting this essential process. ImportanceStaphylococcus aureus is a serious human pathogen and a model organism for cell-division studies in spherical bacteria. We show that SosA is the DNA-damage-inducible cell-division inhibitor in S. aureus that upon expression causes cell swelling and cessation of the cell cycle at a characteristic stage post septum initiation but prior to division plate completion. SosA appears to function via an extracellular activity and is likely to do so by interfering with the essential membrane-associated division proteins, while at the same time being negatively regulated by the membrane protease CtpA. This report represents the first description of the process behind cell-division inhibition in coccoid bacteria. As several pathogens are included in this category, uncovering the molecular details of SosA activity and control can lead to identification of new targets for development of valuable anti-bacterial drugs.

microbiology↗