Ho-Ching Tiffany Tsui

Regulation of the pspA Virulence Factor and Essential pcsB Murein Biosynthetic Genes by the Phosphorylated VicR (YycF) Response Regulator in Streptococcus pneumoniae

The VicRK (YycFG) two-component regulatory system (TCS) is required for virulence of the human respiratory pathogen Streptococcus pneumoniae (pneumococcus). The VicR (YycF) response regulator (RR) is essential through its positive regulation of pcsB, which encodes an extracellular protein that mediates murein biosynthesis. To determine other genes that are regulated by VicR, we performed microarray analyses on a unique DeltavicR deletion mutant, which was constructed by uncoupling regulation of pcsB. Results from these microarray experiments support the idea that the VicR RR exerts strong positive regulation on the transcription of a set of genes encoding important surface proteins, including the PspA virulence factor, two proteins (Spr0096 and Spr1875) containing LysM peptidoglycan-binding domains, and a putative membrane protein (Spr0709) of unknown function. To demonstrate direct regulation, we performed band shift and footprinting experiments using purified unphosphorylated VicR and phosphorylated VicR-P, which was prepared by reaction with acetyl phosphate. VicR and VicR-P bound to regions upstream of pcsB, pspA, spr0096, spr1875, and spr0709. Phosphorylation of VicR to VicR-P increased the apparent strength and changed the nature of binding to these regions. DNase I footprinting of VicR and VicR-P bound to regions upstream of pcsB, pspA, spr0096, and spr1875 showed protection of extended regions containing a degenerate sequence related to a previously proposed consensus. These combined approaches did not support autoregulation of the vicRKX operon or substantive direct regulation of fatty acid biosynthesis by VicR or VicR-P. However, the DeltavicR mutant required fatty acids in some conditions, which supports the notion that the VicRK TCS may mediate membrane integrity as well as murein biosynthesis and virulence factor expression in S. pneumoniae.

A new structural paradigm in copper resistance in Streptococcus pneumoniae

Copper resistance has emerged as an important virulence determinant of microbial pathogens. In Streptococcus pneumoniae, copper resistance is mediated by the copper-responsive repressor CopY, CupA, and CopA, a copper effluxing P1B-type ATPase. We show here that CupA is a novel cell membrane-anchored Cu(I) chaperone, and that a Cu(I)-binding competent, membrane-localized CupA is obligatory for copper resistance. The crystal structures of the soluble domain of CupA (sCupA) and the N-terminal metal binding domain (MBD) of CopA (CopAMBD) reveal isostructural cupredoxin-like folds each harboring a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site on sCupA to the high-affinity site of CopAMBD. However, copper binding by CopAMBD is not essential for cellular copper resistance, consistent with a primary role of CupA in cytoplasmic Cu(I) sequestration and/or direct delivery to the transmembrane site of CopA for cellular efflux.

Dynamic Distribution of the SecA and SecY Translocase Subunits and Septal Localization of the HtrA Surface Chaperone/Protease during Streptococcus pneumoniae D39 Cell Division

ABSTRACT The Sec translocase pathway is the major route for protein transport across and into the cytoplasmic membrane of bacteria. Previous studies reported that the SecA translocase ATP-binding subunit and the cell surface HtrA protease/chaperone formed a single microdomain, termed “ExPortal,” in some species of ellipsoidal (ovococcus) Gram-positive bacteria, including Streptococcus pyogenes. To investigate the generality of microdomain formation, we determined the distribution of SecA and SecY by immunofluorescent microscopy in Streptococcus pneumoniae (pneumococcus), which is an ovococcus species evolutionarily distant from S. pyogenes. In the majority (≥75%) of exponentially growing cells, S. pneumoniae SecA (SecASpn) and SecYSpn located dynamically in cells at different stages of division. In early divisional cells, both Sec subunits concentrated at equators, which are future sites of constriction. Further along in division, SecASpn and SecYSpn remained localized at mid-cell septa. In late divisional cells, both Sec subunits were hemispherically distributed in the regions between septa and the future equators of dividing cells. In contrast, the HtrASpn homologue localized to the equators and septa of most (>90%) dividing cells, whereas the SrtASpn sortase located over the surface of cells in no discernable pattern. This dynamic pattern of Sec distribution was not perturbed by the absence of flotillin family proteins, but was largely absent in most cells in early stationary phase and in ∆cls mutants lacking cardiolipin synthase. These results do not support the existence of an ExPortal microdomain in S. pneumoniae. Instead, the localization of the pneumococcal Sec translocase depends on the stage of cell division and anionic phospholipid content. IMPORTANCE Two patterns of Sec translocase distribution, an ExPortal microdomain in certain ovococcus-shaped species like Streptococcus pyogenes and a spiral pattern in rod-shaped species like Bacillus subtilis, have been reported for Gram-positive bacteria. This study provides evidence for a third pattern of Sec localization in the ovococcus human pathogen Streptococcus pneumoniae. The SecA motor and SecY channel subunits of the Sec translocase localize dynamically to different places in the mid-cell region during the division cycle of exponentially growing, but not stationary-phase, S. pneumoniae. Unexpectedly, the S. pneumoniae HtrA (HtrASpn) protease/chaperone principally localizes to cell equators and division septa. The coincident localization of SecASpn, SecYSpn, and HtrASpn to regions of peptidoglycan (PG) biosynthesis in unstressed, growing cells suggests that the pneumococcal Sec translocase directs assembly of the PG biosynthesis apparatus to regions where it is needed during division and that HtrASpn may play a general role in quality control of proteins exported by the Sec translocase. Two patterns of Sec translocase distribution, an ExPortal microdomain in certain ovococcus-shaped species like Streptococcus pyogenes and a spiral pattern in rod-shaped species like Bacillus subtilis, have been reported for Gram-positive bacteria. This study provides evidence for a third pattern of Sec localization in the ovococcus human pathogen Streptococcus pneumoniae. The SecA motor and SecY channel subunits of the Sec translocase localize dynamically to different places in the mid-cell region during the division cycle of exponentially growing, but not stationary-phase, S. pneumoniae. Unexpectedly, the S. pneumoniae HtrA (HtrASpn) protease/chaperone principally localizes to cell equators and division septa. The coincident localization of SecASpn, SecYSpn, and HtrASpn to regions of peptidoglycan (PG) biosynthesis in unstressed, growing cells suggests that the pneumococcal Sec translocase directs assembly of the PG biosynthesis apparatus to regions where it is needed during division and that HtrASpn may play a general role in quality control of proteins exported by the Sec translocase.

Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39.

In ellipsoid‐shaped ovococcus bacteria, such as the pathogen Streptococcus pneumoniae (pneumococcus), side‐wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin‐binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG‐domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ΔmltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. The ‘synthetic viable’ genetic relationship between Δpbp1a and ΔmltG mutations extends to essential ΔmreCD and ΔrodZ mutations that misregulate peripheral PG synthesis. Remarkably, the single MltG(Y488D) change suppresses the requirement for PBP2b, MreCD, RodZ and RodA. Structural modeling and comparisons, catalytic‐site changes and an interspecies chimera indicate that pneumococcal MltG is the functional homologue of the recently reported MltG endo‐lytic transglycosylase of Escherichia coli. Depletion of pneumococcal MltG or mltG(Y488D) increases sphericity of cells, and MltG localizes with peripheral PG synthesis proteins during division. Finally, growth of Δpbp1a ΔmltG or mltG(Y488D) mutants depends on induction of expression of the WalRK TCS regulon of PG hydrolases. These results fit a model in which MltG releases anchored PG glycan strands synthesized by PBP1a for crosslinking by a PBP2b:RodA complex in peripheral PG synthesis.

Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production in Streptococcus pneumoniae D39

Adaptation to endogenous oxidative stress is an integral aspect of Streptococcus pneumoniae colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H2O2 and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in S. pneumoniae. ABSTRACT The catalase-negative, facultative anaerobe Streptococcus pneumoniae D39 is naturally resistant to hydrogen peroxide (H2O2) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced H2O2. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that ΔlctO mutants produce significantly lower H2O2. In addition, both the SpxB pathway and a candidate pyruvate dehydrogenase complex (PDHC) pathway contribute to acetyl coenzyme A (acetyl-CoA) production during aerobic growth, and the pyruvate format lyase (PFL) pathway is the major acetyl-CoA pathway during anaerobic growth. Microarray analysis of the D39 strain cultured under aerobic versus strict anaerobic conditions shows upregulation of spxB, a gene encoding a rhodanese-like protein (locus tag spd0091), tpxD, sodA, piuB, piuD, and an Fe-S protein biogenesis operon under H2O2-producing conditions. Proteome profiling of H2O2-induced sulfenylation reveals that sulfenylation levels correlate with cellular H2O2 production, with endogenous sulfenylation of ≈50 proteins. Deletion of tpxD increases cellular sulfenylation 5-fold and has an inhibitory effect on ATP generation. Two major targets of protein sulfenylation are glyceraldehyde-3-phosphate dehydrogenase (GapA) and SpxB itself, but targets also include pyruvate kinase, LctO, AdhE, and acetate kinase (AckA). Sulfenylation of GapA is inhibitory, while the effect on SpxB activity is negligible. Strikingly, four enzymes of capsular polysaccharide biosynthesis are sulfenylated, as are enzymes associated with nucleotide biosynthesis via ribulose-5-phosphate. We propose that LctO/SpxB-generated H2O2 functions as a signaling molecule to downregulate capsule production and drive altered flux through sugar utilization pathways. IMPORTANCE Adaptation to endogenous oxidative stress is an integral aspect of Streptococcus pneumoniae colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H2O2 and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in S. pneumoniae.

Absence of the KhpA and KhpB (JAG/EloR) RNA-Binding Proteins Suppresses the Requirement for PBP2b by Overproduction of FtsA in Streptococcus pneumoniae D39

Suppressor mutations were isolated that obviate the requirement for essential PBP2b in peripheral elongation of peptidoglycan from the midcells of dividing Streptococcus pneumoniae D39 background cells. One suppressor was in a gene encoding a single KH‐domain protein (KhpA). ΔkhpA suppresses deletions in most, but not all (mltG), genes involved in peripheral PG synthesis and in the gpsB regulatory gene. ΔkhpA mutations reduce growth rate, decrease cell size, minimally affect shape and induce expression of the WalRK cell‐wall stress regulon. Reciprocal co‐immunoprecipitations show that KhpA forms a complex in cells with another KH‐domain protein (KhpB/JAG/EloR). ΔkhpA and ΔkhpB mutants phenocopy each other exactly, consistent with a direct interaction. RNA‐immunoprecipitation showed that KhpA/KhpB bind an overlapping set of RNAs in cells. Phosphorylation of KhpB reported previously does not affect KhpB function in the D39 progenitor background. A chromosome duplication implicated FtsA overproduction in Δpbp2b suppression. We show that cellular FtsA concentration is negatively regulated by KhpA/B at the post‐transcriptional level and that FtsA overproduction is necessary and sufficient for suppression of Δpbp2b. However, increased FtsA only partially accounts for the phenotypes of ΔkhpA mutants. Together, these results suggest that multimeric KhpA/B may function as a pleiotropic RNA chaperone controlling pneumococcal cell division.