Vijay Pancholi

Role of the C-Terminal Lysine Residues of Streptococcal Surface Enolase in Glu- and Lys-Plasminogen-Binding Activities of Group A Streptococci

ABSTRACT Streptococcal surface enolase (SEN) is a major plasminogen-binding protein of group A streptococci. Our earlier biochemical studies have suggested that the region responsible for this property is likely located at the C-terminal end of the SEN molecule. In the present study, the gene encoding SEN was cloned from group A streptococci M6 isolate D471. A series of mutations in the sen gene corresponding to the C-terminal region (428KSFYNLKK435) of the SEN molecule were created by either deleting one or more terminal lysine residues or replacing them with leucine. All purified recombinant SEN proteins with altered C-terminal ends were found to be enzymatically active and were analyzed for their Glu- and Lys-plasminogen-binding activities. Wild-type SEN bound to Lys-plasminogen with almost three times more affinity than to Glu-plasminogen. However, the recombinant mutant SEN proteins with a deletion of Lys434-435 or with K435L and K434-435L replacements showed a significant decrease in Glu- and Lys-plasminogen-binding activities. Accordingly, a streptococcal mutant expressing SEN-K434-435L showed a significant decrease in Glu- and Lys-plasminogen-binding activities. Biochemical and functional analyses of the isogenic mutant strain revealed a significant decrease in its abilities to cleave a chromogenic tripeptide substrate, acquire plasminogen from human plasma, and penetrate the extracellular matrix. Together, these data indicate that the last two C-terminal lysine residues of surface-exposed SEN contribute significantly to the plasminogen-binding activity of intact group A streptococci and hence to their ability to exploit host properties to their own advantage in tissue invasion.

Modulation of Cell Wall Structure and Antimicrobial Susceptibility by a Staphylococcus aureus Eukaryote-Like Serine/Threonine Kinase and Phosphatase

ABSTRACT It is well established that prokaryotes and eukaryotes alike utilize phosphotransfer to regulate cellular functions. One method by which this occurs is via eukaryote-like serine/threonine kinase (ESTK)- and phosphatase (ESTP)-regulated pathways. The role of these enzymes in Staphylococcus aureus has not yet been examined. This resilient organism is a common cause of hospital-acquired and community-associated infections, infecting immunocompromised and immunocompetent hosts alike. In this study, we have characterized a major functional ESTK (STK) and ESTP (STP) in S. aureus and found them to be critical modulators of cell wall structure and susceptibility to cell wall-acting β-lactam antibiotics. By utilizing gene knockout strategies, we created S. aureus N315 mutants lacking STP and/or STK. The strain lacking both STP and STK displayed notable cell division defects, including multiple and incomplete septa, bulging, and irregular cell size, as observed by transmission electron microscopy. Mutants lacking STP alone displayed thickened cell walls and increased resistance to the peptidoglycan-targeting glycylglycine endopeptidase lysostaphin, compared to the wild type. Additionally, mutant strains lacking STK or both STK and STP displayed increased sensitivity to cell wall-acting cephalosporin and carbapenem antibiotics. Together, these results indicate that S. aureus STK- and STP-mediated reversible phosphorylation reactions play a critical role in proper cell wall architecture, and thus the modulation of antimicrobial resistance, in S. aureus.

Surface-Expressed Enolase Contributes to the Pathogenesis of Clinical Isolate SSU of Aeromonas hydrophila

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latters tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease alpha(2)-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.

Surface Export of GAPDH/SDH, a Glycolytic Enzyme, Is Essential for Streptococcus pyogenes Virulence

ABSTRACT Streptococcal surface dehydrogenase (SDH) (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) is an anchorless major multifunctional surface protein in group A Streptococcus (GAS) with the ability to bind important mammalian proteins, including plasmin(ogen). Although several biological properties of SDH are suggestive of its possible role in GAS virulence, its direct role in GAS pathogenesis has not been ascertained because it is essential for GAS survival. Thus, it has remained enigmatic as to “how and why” SDH/GAPDH is exported onto the bacterial surface. The present investigation highlights “why” SDH is exported onto the GAS surface. Differential microarray-based genome-wide transcript abundance analysis was carried out using a specific mutant, which was created by inserting a hydrophobic tail at the C-terminal end of SDH (M1-SDHHBtail) and thus preventing its exportation onto the GAS surface. This analysis revealed downregulation of the majority of genes involved in GAS virulence and genes belonging to carbohydrate and amino acid metabolism and upregulation of those related to lipid metabolism. The complete attenuation of this mutant for virulence in the mouse model and the decreased and increased virulence of the wild-type and mutant strains postcomplementation with SDHHBtail and SDH, respectively, indicated that the SDH surface export indeed regulates GAS virulence. M1-SDHHBtail also displayed unaltered growth patterns, increased intracellular ATP concentration and Hpr double phosphorylation, and significantly reduced pH tolerance, streptolysin S, and SpeB activities. These phenotypic and physiological changes observed in the mutant despite the unaltered expression levels of established transcriptional regulators further highlight the fact that SDH interfaces with many regulators and its surface exportation is essential for GAS virulence. IMPORTANCE Streptococcal surface dehydrogenase (SDH), a classical anchorless cytoplasmically localized glycolytic enzyme, is exported onto the group A Streptococcus (GAS) surface through a hitherto unknown mechanism(s). It has not been known why GAS or other prokaryotes should export this protein onto the surface. By genetic manipulations, we created a novel GAS mutant strain expressing SDH with a 12-amino-acid hydrophobic tail at its C-terminal end and thus were able to prevent its surface exportation without altering its enzymatic activity or growth pattern. Interestingly, the mutant was completely attenuated for virulence in a mouse peritonitis model. The global gene expression profiles of this mutant reveal that the surface exportation of SDH is mandatory to maintain GAS virulence. The ability of GAS as a successful pathogen to localize SDH in the cytoplasm as well as on the surface is physiologically relevant and dynamically obligatory to fine-tune the functions of many transcriptional regulators and also to exploit its virulence properties for infection. Streptococcal surface dehydrogenase (SDH), a classical anchorless cytoplasmically localized glycolytic enzyme, is exported onto the group A Streptococcus (GAS) surface through a hitherto unknown mechanism(s). It has not been known why GAS or other prokaryotes should export this protein onto the surface. By genetic manipulations, we created a novel GAS mutant strain expressing SDH with a 12-amino-acid hydrophobic tail at its C-terminal end and thus were able to prevent its surface exportation without altering its enzymatic activity or growth pattern. Interestingly, the mutant was completely attenuated for virulence in a mouse peritonitis model. The global gene expression profiles of this mutant reveal that the surface exportation of SDH is mandatory to maintain GAS virulence. The ability of GAS as a successful pathogen to localize SDH in the cytoplasm as well as on the surface is physiologically relevant and dynamically obligatory to fine-tune the functions of many transcriptional regulators and also to exploit its virulence properties for infection.

α‐enolase: a novel autoantigen in patients with premature ovarian failure

Objective  Although controversial, the presence of circulating antiovarian antibodies (AOA) may be considered a marker of autoimmune premature ovarian failure (POF). The purpose of the present work was to evaluate the presence of AOA in POF patients, and to identify a possible autoantigen in order to develop a reliable diagnostic tool that might help to determine the real prevalence of autoimmune POF.

Surface-exposed histone-like protein a modulates adherence of Streptococcus gallolyticus to colon adenocarcinoma cells.

ABSTRACT Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is a low-grade opportunistic pathogen which is considered to be associated with colon cancer. It is thought that colon polyps or tumors are the main portal of entry for this bacterium and that heparan sulfate proteoglycans (HSPGs) at the colon tumor cell surface are involved in bacterial adherence during the first stages of infection. In this study, we have shown that the histone-like protein A (HlpA) of S. gallolyticus is a genuine anchorless bacterial surface protein that binds to lipoteichoic acid (LTA) of the gram-positive cell wall in a growth phase-dependent manner. In addition, HlpA was shown to be one of the major heparin-binding proteins of S. gallolyticus able to bind to the HSPG-expressing colon tumor cell lines HCT116 and HT-29. Strikingly, although wild-type levels of HlpA appeared to contribute to adherence, coating of additional HlpA at the bacterial surface resulted in reduced binding to colon tumor cells. This may be explained by the fact that heparan sulfate and LTA compete for the same binding site in HlpA. Altogether, this study implies that HlpA serves as a fine-tuning factor for bacterial adherence.

Role of Serine/Threonine Phosphatase (SP-STP) in Streptococcus pyogenes Physiology and Virulence

Background: Unlike for eukaryote-type serine/threonine kinase of group A Streptococcus (GAS), significance of its cognate serine/threonine phosphatase (SP-STP) remains elusive. Results: SP-STP is crucial for GAS pathophysiology. Conclusion: SP-STP is not essential for GAS survival, but its optimal concentration is critical for cognately maintained homeostasis within GAS. Significance: This work opens up avenues to understand the role of secretory SP-STP as an important virulence determinant in the host. Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.

The Peptidoglycan-Associated Lipoprotein OprL Helps Protect a Pseudomonas aeruginosa Mutant Devoid of the Transactivator OxyR from Hydrogen Peroxide-Mediated Killing during Planktonic and Biofilm Culture

OxyR controls H(2)O(2)-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<10(7)/ml) organisms are easily killed by micromolar H(2)O(2). The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H(2)O(2). We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H(2)O(2) than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H(2)O(2). Given the sensitivity of oxyR oprL bacteria to planktonic H(2)O(2), we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H(2)O(2)-mediated killing. Surprisingly, biofilm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H(2)O(2). Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H(2)O(2) in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.

Strain-Specific Regulatory Role of Eukaryote-Like Serine/Threonine Phosphatase in Pneumococcal Adherence

ABSTRACT Streptococcus pneumoniae exploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase of S. pneumoniae (StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerless phpP knockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknown in vitro and in vivo evidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpP mutants. In particular, StkP (threonine)-phosphorylated RR06 bound to the cbpA promoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.

Serine/Threonine Phosphatase (SP-STP), Secreted from Streptococcus pyogenes, Is a Pro-apoptotic Protein

Background: Eukaryote-type serine/threonine phosphatase (SP-STP) of group A Streptococcus (GAS) is essential for virulence, but its direct role in causing pathological effects in the host remains unknown. Results: SP-STP crosses two membrane barriers and causes apoptosis of human pharyngeal cells from within and without. Conclusion: SP-STP is a quintessential virulence-controlling factor. Significance: The study illustrates how GAS utilizes SP-STP to exploit host machinery for its own advantage. This investigation illustrates an important property of eukaryote-type serine/threonine phosphatase (SP-STP) of group A Streptococcus (GAS) in causing programmed cell death of human pharyngeal cells. The secretory nature of SP-STP, its elevated expression in the intracellular GAS, and the ability of wild-type GAS but not the GAS mutant devoid of SP-STP to cause apoptosis of the host cell both in vitro and in vivo suggest that GAS deploys SP-STP as an important virulence determinant to exploit host cell machinery for its own advantage during infection. The exogenously added SP-STP is able to enter the cytoplasm and subsequently traverses into the nucleus in a temporal fashion to cause apoptosis of the pharyngeal cells. The programmed cell death induced by SP-STP, which requires active transcription and de novo protein synthesis, is also caspase-dependent. Furthermore, the entry of SP-STP into the cytoplasm is dependent on its secondary structure as the catalytically inactive SP-STP with an altered structure is unable to internalize and cause apoptosis. The ectopically expressed wild-type SP-STP was found to be in the nucleus and conferred apoptosis of Detroit 562 pharyngeal cells. However, the catalytically inactive SP-STP was unable to cause apoptosis even when intracellularly expressed. The ability of SP-STP to activate pro-apoptotic signaling cascades both in the cytoplasm and in the nucleus resulted in mitochondrial dysfunctioning and perturbation in the phosphorylation status of histones in the nucleus. SP-STP thus not only functions as a virulence regulator but also as an important factor responsible for host-related pathogenesis.