Kinetic Characterization of the WalRKSpn (VicRK) Two-Component System of Streptococcus pneumoniae: Dependence of WalKSpn (VicK) Phosphatase Activity on Its PAS Domain▿ †

The WalRK two-component system plays important roles in maintaining cell wall homeostasis and responding to antibiotic stress in low-GC Gram-positive bacteria. In the major human pathogen, Streptococcus pneumoniae, phosphorylated WalRSpn (VicR) response regulator positively controls the transcription of genes encoding the essential PcsB division protein and surface virulence factors. WalRSpn is phosphorylated by the WalKSpn (VicK) histidine kinase. Little is known about the signals sensed by WalK histidine kinases. To gain information about WalKSpn signal transduction, we performed a kinetic characterization of the WalRKSpn autophosphorylation, phosphoryltransferase, and phosphatase reactions. We were unable to purify soluble full-length WalKSpn. Consequently, these analyses were performed using two truncated versions of WalKSpn lacking its single transmembrane domain. The longer version (Δ35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Δ195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Δ35 and Δ195 WalKSpn were similar [Km(ATP) ≈ 37 μM; kcat ≈ 0.10 min−1] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalKSpn∼P were also similar in the phosphoryltransfer reaction to full-length WalRSpn. In contrast, absence of the HAMP-PAS domains significantly diminished the phosphatase activity of WalKSpn for WalRSpn∼P. Deletion and point mutations confirmed that optimal WalKSpn phosphatase activity depended on the PAS domain as well as residues in the DHp domain. In addition, these WalKSpn DHp domain and ΔPAS mutations led to attenuation of virulence in a murine pneumonia model

Regulation of the cell division hydrolase RipC by the FtsEX system in Mycobacterium tuberculosis

15 pags., 9 figs.The FtsEX complex regulates, directly or via a protein mediator depending on bacterial genera, peptidoglycan degradation for cell division. In mycobacteria and Gram-positive bacteria, the FtsEX system directly activates peptidoglycan-hydrolases by a mechanism that remains unclear. Here we report our investigation of Mycobacterium tuberculosis FtsEX as a non-canonical regulator with high basal ATPase activity. The cryo-EM structures of the FtsEX system alone and in complex with RipC, as well as the ATP-activated state, unveil detailed information on the signal transduction mechanism, leading to the activation of RipC. Our findings indicate that RipC is recognized through a "Match and Fit" mechanism, resulting in an asymmetric rearrangement of the extracellular domains of FtsX and a unique inclined binding mode of RipC. This study provides insights into the molecular mechanisms of FtsEX and RipC regulation in the context of a critical human pathogen, guiding the design of drugs targeting peptidoglycan remodeling.This work is supported by grants from the National University of Singapore Start-up grant (the Ministry of Education Academic Research (MOE) Tier 1 Grants 21-0053-A0001, 22-3449-A0001, and 22-3448-A0001 to M.L., NUHSRO/2017/070/SU/01 to L.-T.S.), and the National Research Foundation Fellowship (NRF) (NRFF11-2019-0005 to L.-T.S.), the MOE Tier 2 Fund (MOE-T2EP30222-0015 to M.L., MOE-T2EP30220-0012 to L.-T.S.). In Spain work was supported by grants PID2020-115331GB-100 funded by MCIN/AEI/10.13039/ 501100011033 and CRSII5_198737/1 (Swiss National Science Foundation) to J.A.H.Peer reviewe

Cell envelope defects of different capsule-null mutants in K1 hypervirulent Klebsiella pneumoniae can affect bacterial pathogenesis

10.1111/mmi.14447MOLECULAR MICROBIOLOGY1135889-90

The divisome but not the elongasome organizes capsule synthesis in Streptococcus pneumoniae

Abstract The bacterial cell envelope consists of multiple layers, including the peptidoglycan cell wall, one or two membranes, and often an external layer composed of capsular polysaccharides (CPS) or other components. How the synthesis of all these layers is precisely coordinated remains unclear. Here, we identify a mechanism that coordinates the synthesis of CPS and peptidoglycan in Streptococcus pneumoniae. We show that CPS synthesis initiates from the division septum and propagates along the long axis of the cell, organized by the tyrosine kinase system CpsCD. CpsC and the rest of the CPS synthesis complex are recruited to the septum by proteins associated with the divisome (a complex involved in septal peptidoglycan synthesis) but not the elongasome (involved in peripheral peptidoglycan synthesis). Assembly of the CPS complex starts with CpsCD, then CpsA and CpsH, the glycosyltransferases, and finally CpsJ. Remarkably, targeting CpsC to the cell pole is sufficient to reposition CPS synthesis, leading to diplococci that lack CPS at the septum. We propose that septal CPS synthesis is important for chain formation and complement evasion, thereby promoting bacterial survival inside the host

RNA thermosensors facilitate Streptococcus pneumoniae and Haemophilus influenzae immune evasion

10.1371/journal.ppat.1009513PLoS Pathogens174e100951

High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in Streptococcus pneumoniae.

10.1128/mBio.02615-21mBioe0261521

Decoding capsule synthesis in Streptococcuspneumoniae

10.1093/femsre/fuaa067FEMS Microbiology Reviews1-1

SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by <i>Streptococcus pneumoniae</i> and <i>Staphylococcus aureus</i>

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike–biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients

MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis

Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ(M) and cells lacking MurJBs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition