Determinants of pneumococcal adhesion to human alveolar epithelial cells are identified at both the serotype and genotype levels and a regulatory node is revealed that links two signaling systems to a single virulence output is revealed.
Abstract
Streptococcus pneumoniae establishes asymptomatic nasopharyngeal colonization before progressing to invasive disease, a process that requires precise spatiotemporal regulation of surface adhesins. Using a barcoded capsule-switch mutant library and randomly barcoded transposon sequencing (RB-TnSeq), we identified determinants of pneumococcal adhesion to the human alveolar epithelial cell line A549. The capsule masked surface adhesins and reduced binding across all 84 serotypes tested. The transposon screen revealed that overexpression of vncS, the sensor kinase of the two-component system TCS10, markedly increased adhesion, whereas disruption of its cognate response regulator VncR had no detectable effect. Transcriptomic profiling and genetic epistasis established that the adhesin CbpA, known to be regulated by TCS06, is the effector of VncS-driven adhesion. VncS activated cbpA expression through the non-cognate response regulator RR06, independently of VncR. Phos-tag gel analysis demonstrated that VncS cross-phosphorylates RR06 in vivo, and that the cognate kinase HK06 limits this crosstalk by acting primarily as a phosphatase toward RR06. Substitution of the conserved S241 residue of HK06 increased RR06∼P levels and upregulated cbpA. By contrast, deleting vncS and hk06 reduced RR06 phosphorylation to nearly undetectable levels. These findings resolve a long-standing mechanistic controversy regarding RR06 activation and uncover a potentially unrecognized role for VncS in regulating pneumococcal host cell adhesion. SIGNIFICANCE Streptococcus pneumoniae is a leading cause of pneumonia, and its ability to colonize the human airway depends on surface adhesins. Several two-component signaling systems are known to regulate adhesin expression, but how their outputs are integrated remains incompletely understood. Here, we identify determinants of pneumococcal adhesion to human alveolar epithelial cells at both the serotype and genotype levels. We show that the histidine kinase VncS cross-phosphorylates the non-cognate response regulator RR06 to activate the adhesin CbpA, whereas the cognate kinase HK06 primarily acts as a phosphatase to restrain this crosstalk. These findings resolve a long-standing controversy regarding RR06 activation and reveal a regulatory node that links two signaling systems to a single virulence output.
Deletion of fabR reduced host cell adherence, increased serum sensitivity, enhanced IgG deposition, and decreased vitronectin binding compared with the wild-type strain, and ΔfabR mutants from multiple NTHi strains showed increased metabolic activity, indicating a conserved role for FabR in metabolic regulation.
Martina Janoušková, Yu-Ching Su, Sandra Jonsson et al.· Journal of Infectious Diseas...· 0 citations
The proliferation of multidrug-resistant
Klebsiella pneumoniae
strains underscores the urgent need to decipher the transcriptional mechanisms governing their pathogenicity and resistance. This study characterizes the genome-wide regulatory architecture of
K. pneumoniae
MGH 78578 by mapping the binding landscapes of the housekeeping sigma factor RpoD and the general stress response sigma factor RpoS using high-resolution chromatin immunoprecipitation with exonuclease treatment (ChIP-exo). Contrasting with prior low-resolution studies, RpoD was identified as the dominant transcriptional orchestrator, encompassing a larger sigmulon than RpoS across both mid-exponential and stationary phases. While RpoS exhibited a high number of binding sites, it displayed relaxed promoter specificity characterized by the absence of a conserved −35 motif, reflecting a poised and plastic regulatory potential. A distinct evolutionary signature was observed on plasmids, where sigma factor binding density matched or exceeded that of the chromosome, but a smaller proportion of these binding events occurred at regulatory positions, indicating that these horizontally acquired sequences are not yet fully adapted to the host’s sigma factor recognition patterns. In the context of pathogenicity, distinct binding patterns were observed: RpoD was predominantly associated with core antimicrobial resistance determinants, including plasmid-encoded β-lactamases, whereas RpoS was preferentially linked to surface-remodeling virulence factors. These findings provide a high-resolution reconstruction of the
K. pneumoniae
transcriptional network, revealing how evolutionary history and sigma factor specificity coordinate the complex interplay between multidrug resistance and virulence.
Minchang Jang, Zoe K. Scott-Nevros, Sohee Yoon et al.· Biotechnology and Bioprocess...· 0 citations
Uropathogenic Escherichia coli (UPEC) remains the most common etiologic agent of community-acquired urinary tract infections (UTIs), largely due to its extensive virulence arsenal and adaptive regulatory mechanisms. In this study, we examined the phenotypic regulation of major virulence factors—hemolysin production, biofilm, swarming motility, and adhesion—under exposure to sub-inhibitory concentrations of Ceftazidime, an extended-spectrum β-lactam antibiotic. The transcriptional response of the BarA/UvrY two-component regulatory system was also examined to unravel its role in response to antibiotic stress. Using an extensive range of microbiological, molecular, and phenotypic tests, we unveiled differential profiles of virulence traits. Exposure to sub-MICs Ceftazidime markedly diminished hemolytic activity, biofilm formation, and adhesive capacity, with swarming motility exhibiting almost complete inhibition. Moreover, quantitative real-time PCR (qRT-PCR) analysis revealed variable transcriptional responses of BarA and UvrY in the tested isolates, with the recovery of BarA expression noted after prolonged exposure to antibiotics. These findings emphasize the dynamic expression of UPEC's stress response mechanisms and outline the central role of Bar/UvrY in modulating pathoadaptive mechanisms under antibiotic stress.
Ali Hazim Jabbar, M. Flayyih· Iraqi Journal of Science· 0 citations
MFSD6 is a newly identified receptor that mediates the invasion of respiratory cells by enterovirus D68 (EV-D68), a non-polio enterovirus that causes severe respiratory disease and poliomyelitis-like illness in children. Here, we report near-atomic-resolution cryo-electron microscopy (cryo-EM) structures of historical and contemporary AFM-associated EV-D68 strains, together with their complexes bound to the third extracellular loop of MFSD6 (MFSD6-L3). These structures uncover a previously unrecognized “binary gating switch” mechanism of virus-receptor engagement that differs from the reported model. In this mechanism, the N200-V208 segment of MFSD6, carrying a glycosylated Asn207, inserts into one capsomer, whereas the sialyl-Gal-terminated glycan of MFSD6 engages an adjacent capsomer. Neu5Ac binding induces conformational rearrangements that expel the pocket factor, destabilize the virion, and prime infection. Functional analyses further define the contributions of the receptor-contacting residues and glycans to viral attachment and entry. Together, our findings refine the molecular basis of EV-D68 recognition of MFSD6 and reveal a glycan-mediated dual-lock mechanism that may enhance receptor specificity, prevent premature uncoating, and ensure productive infection only upon engagement of the correct host receptor. These results provide broader insight into enterovirus tropism and establish a framework for structure-guided antiviral design. Liang et al. show how enterovirus D68 uses the respiratory receptor MFSD6 and sugar modifications to enter cells, revealing a checkpoint that primes the virus for infection.
Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.
Eunjeong Lee, Blaine H. Gordon, J. Redzic et al.· Biomolecules· 0 citations
ABSTRACT Group A Streptococcus (GAS) is a human-restricted pathogen whose global incidence has surged in the post-COVID era. The ability of GAS to shift from a colonizing to invasive phenotype depends on coordinated virulence gene regulation in response to host-derived signals. However, the mechanisms by which individual stress-sensing systems interact to reshape the virulence gene regulatory landscape remain incompletely understood. Here, we define the regulatory programs of two conserved transcriptional regulator paralogs, SpxA1 and SpxA2, using an integrated multi-omic approach combining RNA-seq, data-independent acquisition proteomics, NanoString-based transcriptional profiling across multiple host-relevant stress conditions, and chromatin immunoprecipitation with exonuclease treatment (ChIP-exo). RNA-seq revealed functionally distinct regulons with SpxA1 governing oxidative stress defense and SpxA2 coordinating virulence-associated gene expression linked to the CovRS two-component regulatory system. Proteomic analysis established SpxA2 as a ClpXP protease substrate in GAS and identified reciprocal paralog accumulation upon loss of either SpxA1 or SpxA2, consistent with compensatory transcriptional upregulation. NanoString profiling under bacitracin and human neutrophil peptide-1 challenge identified four gene modules with distinct stoichiometry-dependent and condition-dependent regulatory logic, revealing that the SpxA1/SpxA2 ratio rather than the activity of either paralog alone determines which transcriptional programs are engaged. ChIP-exo demonstrated that SpxA2 directly modulates CovR-DNA binding occupancy in a CovR-binding motif-dependent manner, simultaneously antagonizing CovR dimer binding at an extended (25 bp) CovR motif and facilitating CovR monomer binding at the canonical ATTARA motif. These findings establish the LiaFSR-SpxA2-CovRS axis as a cross-regulatory circuit through which GAS cell envelope stress sensing is directly transduced into coordinated virulence gene regulatory changes. IMPORTANCE Group A Streptococcus (GAS) causes millions of infections annually, including a recent global surge in invasive disease. To survive in the human host, GAS must rapidly reprogram virulence gene expression in response to host-derived stresses. This study characterizes two conserved transcriptional regulators, SpxA1 and SpxA2, that govern this response through interaction with RNA polymerase to indirectly influence the DNA-binding activity of downstream transcription factors. We show that SpxA2, activated by a cell envelope stress-sensing system responding to human antimicrobial peptides, reshapes the binding of the master virulence regulator CovR in a promoter-specific manner, coupling cell envelope stress sensing to virulence gene regulation. The stoichiometric balance between SpxA1 and SpxA2 functions as a regulatory rheostat calibrating overall virulence gene regulatory tone, providing a framework for understanding how RNA polymerase-interacting regulators coordinate stress responses and virulence gene control across Gram-positive bacterial pathogens. Group A Streptococcus (GAS) causes millions of infections annually, including a recent global surge in invasive disease. To survive in the human host, GAS must rapidly reprogram virulence gene expression in response to host-derived stresses. This study characterizes two conserved transcriptional regulators, SpxA1 and SpxA2, that govern this response through interaction with RNA polymerase to indirectly influence the DNA-binding activity of downstream transcription factors. We show that SpxA2, activated by a cell envelope stress-sensing system responding to human antimicrobial peptides, reshapes the binding of the master virulence regulator CovR in a promoter-specific manner, coupling cell envelope stress sensing to virulence gene regulation. The stoichiometric balance between SpxA1 and SpxA2 functions as a regulatory rheostat calibrating overall virulence gene regulatory tone, providing a framework for understanding how RNA polymerase-interacting regulators coordinate stress responses and virulence gene control across Gram-positive bacterial pathogens.
Gretchen A. Morrison, L. Vega, Misú Sanson Iglesias et al.· bioRxiv· 0 citations