Biology subjects

Zik, J. J.

Publications and source records attributed to Zik, J. J..

4 recordsLinked to original sources

bioRxiv2026

Cross-phosphorylation of RR06 by the non-cognate kinase VncS activates the adhesin CbpA in Streptococcus pneumoniae.

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. SIGNIFICANCEStreptococcus 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.

microbiology↗

bioRxiv2026

The WalRK two-component system in Streptococcus pneumoniae ensures robustness of secondary wall polymer attachment

Capsular polysaccharide (CPS) is essential for Streptococcus pneumoniae virulence. Yet, the mechanism linking CPS to peptidoglycan (PG) remains unclear. Here, we identified a strong negative genetic interaction between the genes encoding the putative capsule ligase CpsA and the WalK histidine kinase, a component of the WalRK two-component system regulating cell wall homeostasis. In the absence of cpsA, capsule polymers compete with wall teichoic acids for ligase activity to PG. This induces cell wall stress and is sensed by the WalRK system. Overexpression of the PG hydrolase pcsB or disruption of the PG-modifying enzymes pgdA and oatA restored growth of strains lacking cpsA and walK. Furthermore, CpsA overproduction compensates for the loss of other LytR-Cps2A-Psr (LCP) ligases, suggesting it can support capsule and wall teichoic acid syntheses. These findings support the model that LCP ligases are semi-redundant, although they may install secondary polymers on a different residue of PG. This work also suggests that WalRK signaling compensates for reduced capsule and WTA attachment by positively regulating PG hydrolases. SIGNIFICANT STATEMENTStreptococcus pneumoniae causes approximately half a million deaths annually. A powerful public health tool for controlling pneumococcal infections is vaccination against the protective capsule. Yet, the mechanisms by which the capsule layer attaches to the underlying cell wall remain poorly defined. This study shows that the conserved capsule gene CpsA is not strictly required for capsule attachment but instead works together with other LytRIZCpsAIZPsr (LCP) ligases. However, it requires the essential WalRK signaling system to maintain cell envelope integrity. Defects in LCP activity are alleviated by WalRKIZdriven upregulation of peptidoglycan hydrolases, overexpression of PcsB, or inactivating peptidoglycan modifications that limit hydrolysis. These findings reveal coordination among flux to capsule synthesis, secondary wall polymer attachment, and cell wall remodeling. TEASERThe WalRK two-component system responds to cell envelope stress caused by reduced LCP ligase activity.

microbiology↗

bioRxiv2024

Dual transposon sequencing (Dual Tn-seq) to probe genome-wide genetic interactions

Understanding gene function on a genome-wide scale is a fundamental goal in biology. With the advent of next-generation sequencing, high-throughput methods such as transposon sequencing (Tn- seq) were developed to measure fitness of single deletion mutants en masse. Nevertheless, gene redundancy complicates these approaches, as inactivating genes individually may not produce discernable phenotypes. Here, we report Dual Tn-seq, a technique for simultaneously assaying fitness of a comprehensive pool of double mutants. Dual Tn-seq couples random barcode transposon-site sequencing (RB Tn-seq) with the Cre-lox system, allowing us to determine the fitness of {approx}68% of all possible double mutant combinations in the human pathogen Streptococcus pneumoniae. Genetic interactions identified from {approx}1.4 billion double mutants uncovered new avenues in widely conserved biochemical pathways, exemplified by the discovery of a new CTP synthase and a regulator of peptidoglycan biosynthesis. Since Dual Tn-seq has very few requirements, it can be readily adapted to diverse organisms.

microbiology↗

bioRxiv2022

Caulobacter requires anionic sphingolipids and deactivation of fur to lose lipid A

Lipid A, the membrane-anchored portion of lipopolysaccharide, is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here, we identify regulatory and structural factors that together permit Caulobacter crescentus to eliminate lipid A from its OM. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog which we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to eliminate lipid A. Fitness of lipid A-deficient Caulobacter requires a previously uncharacterized anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

microbiology↗