Biology subjects

Veening, J.-W.

Publications and source records attributed to Veening, J.-W..

11 recordsLinked to original sources

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↗

bioRxiv2018

RocS drives chromosome segregation and nucleoid occlusion in Streptococcus pneumoniae

Segregation of replicated chromosomes in bacteria is poorly understood outside some prominent model strains and even less is known about how it is coordinated with other cellular processes. Here we report that RocS is crucial for chromosome segregation in the opportunistic human pathogen Streptococcus pneumoniae. RocS is membrane-bound and interacts both with DNA and the chromosome partitioning protein ParB to properly segregate the origin of replication region to new daughter cells. In addition, we show that RocS interacts with the tyrosine-autokinase CpsD required for polysaccharide capsule biogenesis, which is crucial for S. pneumoniaes ability to prevent host immune detection. Altering the RocS-CpsD interaction drastically hinders chromosome partitioning and cell division. Altogether, this work reveals that RocS is the cornerstone of an atypical nucleoid occlusion system ensuring proper cell division in coordination with the biogenesis of a protective capsular layer.

microbiology↗

bioRxiv2018

The ClpX chaperone controls the Staphylococcus aureus cell cycle but can be bypassed by β-lactam antibiotics

The worldwide spread of Staphylococcus aureus strains resistant to almost all {beta}-lactam antibiotics is of major clinical concern. {beta}-lactams interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Here we show that sub-lethal doses of {beta}-lactams stimulate the growth of S. aureus mutants lacking the widely conserved chaperone ClpX. S. aureus clpX mutants have a severe growth defect at temperatures below 37{degrees}C, and we reasoned that a better understanding of this growth defect could provide novel insights into how {beta}-lactam antibiotics interfere with growth of S. aureus. We demonstrate that ClpX is important for coordinating the S. aureus cell cycle, and that S. aureus cells devoid of ClpX fail to divide, or lyze spontaneously, at high frequency unless {beta}-lactams are added to the growth medium. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30{degrees}C, but not at 37{degrees}C. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, {beta}-lactams restored septum synthesis and prevented premature autolytic splitting of clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with {beta}-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, underscoring a functional link between the PBP activity and WTA biosynthesis. The finding that {beta} -lactams can prevent lysis and restore septum synthesis of a mutant with dysregulated cell division lends support to the idea that PBPs function as coordinators of cell division and that {beta} -lactams do not kill S. aureus simply by weakening the cell wall.\n\nAuthor SummaryThe bacterium Staphylococcus aureus is a major cause of human disease, and the rapid spread of S. aureus strains that are resistant to almost all {beta}-lactam antibiotics has made treatment increasingly difficult. {beta}-lactams interfere with cross-linking of the bacterial cell wall but the killing mechanism of this important class of antibiotics is still not fully understood. Here we provide novel insight into this topic by examining a defined S. aureus mutant that has the unusual property of growing markedly better in the presence of {beta}-lactams. Without {beta}-lactams this mutant dies spontaneously at a high frequency due to premature separation of daughter cells during cell division. Cell death of the mutant can, however, be prevented either by exposure to {beta}-lactam antibiotics or by inhibiting synthesis of wall teichoic acid, a major component of the cell wall in Gram-positive bacteria with a conserved role in activation of autolytic splitting of daughter cells. The finding that the detrimental effect of {beta}-lactam antibiotics can be reversed by a mutation that affect the coordination of cell division emphasizes the idea that {beta}-lactams do not kill S. aureus simply by weakening the cell wall but rather by interference with the coordination of cell division.

microbiology↗

bioRxiv2018

Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39

A precise understanding of the genomic organization into transcriptional units and their regulation is essential for our comprehension of opportunistic human pathogens and how they cause disease. Using single-molecule real-time (PacBio) sequencing we unambiguously determined the genome sequence of Streptococcus pneumoniae strain D39 and revealed several inversions previously undetected by short-read sequencing. Significantly, a chromosomal inversion results in antigenic variation of PhtD, an important surface-exposed virulence factor. We generated a new genome annotation using automated tools, followed by manual curation, reflecting the current knowledge in the field. By combining sequence-driven terminator prediction, deep paired-end transcriptome sequencing and enrichment of primary transcripts by Cappable-Seq, we mapped 1,015 transcriptional start sites and 748 termination sites. Using this new genomic map, we identified several new small RNAs (sRNAs), riboswitches (including twelve previously misidentified as sRNAs), and antisense RNAs. In total, we annotated 92 new protein-encoding genes, 39 sRNAs and 165 pseudogenes, bringing the S. pneumoniae D39 repertoire to 2,151 genetic elements. We report operon structures and observed that 9% of operons lack a 5-UTR. The genome data is accessible in an online resource called PneumoBrowse (https://veeninglab.com/pneumobrowse) providing one of the most complete inventories of a bacterial genome to date. PneumoBrowse will accelerate pneumococcal research and the development of new prevention and treatment strategies.

microbiology↗

bioRxiv2018

High-resolution analysis of the pneumococcal transcriptome under a wide range of infection-relevant conditions

Streptococcus pneumoniae is an opportunistic human pathogen that typically colonizes the nasopharyngeal passage and causes lethal disease in other host niches such as the lung or the meninges. How pneumococcal genes are expressed and regulated at the different stages of its life cycle, as commensal or as pathogen, has not been entirely described. To chart the transcriptional responses of S. pneumoniae, we quantified the transcriptome under 22 different infection-relevant conditions. The transcriptomic compendium exposed a high level of dynamic expression and, strikingly, all annotated pneumococcal genomic features were expressed in at least one of the studied conditions. By computing the correlation of gene expression of every two genes across all studied conditions, we created a co-expression matrix that provides valuable information on both operon structure and regulatory processes. The co-expression data is highly consistent with well-characterized operons and regulons, such as the PyrR, ComE and ComX regulons, and has allowed us to identify a new member of the competence regulon. Finally, we created an interactive data center named PneumoExpress (www.veeninglab.com/pneumoexpress) that enables users to access the expression data as well as the co-expression matrix in an intuitive and efficient manner, providing a valuable resource to the pneumococcal research community.

microbiology↗

bioRxiv2018

Antibiotic-induced cell chaining triggers pneumococcal competence by reshaping quorum sensing to autocrine signaling

Streptococcus pneumoniae can acquire antibiotic resistance by activation of competence and subsequent DNA uptake. Several antibiotics induce competence by disrupting protein-quality control or perturbing DNA replication. Here, we demonstrate that aztreonam (AZT) and clavulanic acid (CLA) also promote competence. We show that both compounds induce cell chain formation by targeting the D,D-carboxypeptidase PBP3. In support of the hypothesis that chain formation promotes competence, we demonstrate that an autolysin mutant (lytB) is hypercompetent. As competence is initiated by the binding of a small extracellular peptide (CSP) to a membrane-anchored receptor (ComD), we wondered if chain formation alters CSP diffusion and thereby sensing by ComD. Indeed, the presence of AZT or CLA affects competence synchronization by switching CSP-based quorum sensing to autocrine-like signaling, as CSP is retained to chained cells and no longer shared in a common pool. Together, these insights demonstrate the versatility of quorum sensing in integrating different stresses and highlight that certain antibiotics should be prescribed with care not to drive the spread of antibiotic resistance.

microbiology↗

bioRxiv2018

CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus

Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozESpn of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the {Delta}cozEa{Delta}cozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the {Delta}cozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with the cell division protein EzrA, and that EzrA-GFP mislocalizes in the absence of CozEa and CozEb. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozESpn-depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.

microbiology↗

bioRxiv2018

The Streptococcus pneumoniae Competence-Induced BriC Peptide Promotes Nasopharyngeal Colonization and Impacts Biofilm Development

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

microbiology↗

bioRxiv2016

High-throughput CRISPRi phenotyping in Streptococcus pneumoniae identifies new essential genes involved in cell wall synthesis and competence development

Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae. However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon-sequencing (Tn-Seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD1416 and SPD1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD1198 and SPD1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clpproteolytic system in regulation of competence development and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.

microbiology↗

bioRxiv2016

Chromosome segregation drives division site selection in Streptococcus pneumoniae

Accurate spatial and temporal positioning of the tubulin-like protein FtsZ is key for proper bacterial cell division. Streptococcus pneumoniae (pneumococcus) is an oval-shaped, symmetrically dividing human pathogen lacking the canonical systems for division site control (nucleoid occlusion and the Min-system). Recently, the early division protein MapZ was identified and implicated in pneumococcal division site selection. We show that MapZ is important for proper division plane selection; thus the question remains what drives pneumococcal division site selection. By mapping the cell cycle in detail, we show that directly after replication both chromosomal origin regions localize to the future cell division sites, prior to FtsZ. Perturbing the longitudinal chromosomal organization by mutating the condensin SMC, by CRISPR/Cas9-mediated chromosome cutting or by poisoning DNA decatenation resulted in mistiming of MapZ and FtsZ positioning and subsequent cell elongation. Together, we demonstrate an intimate relationship between DNA replication, chromosome segregation and division site selection in the pneumococcus, providing a simple way to ensure equally sized daughter cells without the necessity for additional protein factors.

microbiology↗

bioRxiv2016

Crosstalk and eavesdropping among quorum sensing peptide signals that regulate bacteriocin production in Streptococcus pneumoniae

Quorum sensing (QS), where bacteria secrete and respond to chemical signals to coordinate population-wide behaviors, has revealed that bacteria are highly social. Here, we investigate how diversity in QS signals and receptors can modify social interactions controlled by the QS system regulating bacteriocin secretion in Streptococcus pneumoniae, encoded by the blp operon (bacteriocin-like peptide). Analysis of 4 096 pneumococcal genomes detected nine blp QS signals (BlpC) and five QS receptor groups (BlpH). Imperfect concordance between signals and receptors suggested widespread social interactions between cells, specifically eavesdropping (where cells respond to signals that they do not produce) and crosstalk (where cells produce signals that non-clones detect). This was confirmed in vitro by measuring the response of reporter strains containing each of six different blp QS receptors to cognate and non-cognate peptides. Assays between pneumococcal colonies grown adjacent to one another provided further evidence that crosstalk and eavesdropping occur at endogenous levels of signal secretion. Finally, simulations of QS strains producing bacteriocins revealed that eavesdropping can be evolutionarily beneficial even when the affinity for non-cognate signals is very weak. Our results highlight that social interactions can mediate intraspecific competition among bacteria and reveal that competitive interactions can be modified by polymorphic QS systems.

microbiology↗