In vitro characterization and identification of potential substrates of a low molecular weight protein tyrosine phosphatase in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen responsible for significant mortality and morbidity worldwide. Within the annotated genome of the pneumococcus lies a previously uncharacterized protein tyrosine phosphatase which shows homology to low molecular weight protein tyrosine phosphatases (LMWPTPs). LMWPTPs modulate many processes critical for the pathogenicity of a number of bacteria including capsular polysaccharide biosynthesis, stress response and persistence in host macrophages. Here, we demonstrate that Spd1837 is indeed a LMWPTP, by purifying the protein, and characterizing its phosphatase activity. Spd1837 showed specific tyrosine phosphatase activity, and it did not form higher order oligomers in contrast to many other LMWPTPs. Substrate-trapping assays using the wild-type and the phosphatase-deficient Spd1837 identified potential substrates/interacting proteins including major metabolic enzymes such as ATP-dependent-6-phosphofructokinase and Hpr kinase/phosphorylase. Given the tight association between the bacterial basic physiology and virulence, this study hopes to prompt further investigation of how the pneumococcus controls its metabolic flux via the LMWPTP Spd1837.

Topology of Streptococcus pneumoniae CpsC, a polysaccharide copolymerase and bacterial protein tyrosine kinase adaptor protein.

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. In Streptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein in S. pneumoniae strain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein's topology was not as predicted. Similar results were seen with membranes from S. pneumoniae strain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.

Tyrosine phosphorylation enhances activity of pneumococcal autolysin LytA.

Tyrosine phosphorylation has long been recognized as a crucial post-translational regulatory mechanism in eukaryotes. However, only in the past decade has recognition been given to the crucial importance of bacterial tyrosine phosphorylation as an important regulatory feature of pathogenesis. This study describes the effect of tyrosine phosphorylation on the activity of a major virulence factor of the pneumococcus, the autolysin LytA, and a possible connection to the Streptococcus pneumoniae capsule synthesis regulatory proteins (CpsB, CpsC and CpsD). We show that in vitro pneumococcal tyrosine kinase, CpsD, and the protein tyrosine phosphatase, CpsB, act to phosphorylate and dephosphorylate LytA. Furthermore, this modulates LytA function in vitro with phosphorylated LytA binding more strongly to the choline analogue DEAE. A phospho-mimetic (Y264E) mutation of the LytA phosphorylation site displayed similar phenotypes as well as an enhanced dimerization capacity. Similarly, tyrosine phosphorylation increased LytA amidase activity, as evidenced by a turbidometric amidase activity assay. Similarly, when the phospho-mimetic mutation was introduced in the chromosomal lytA of S. pneumoniae, autolysis occurred earlier and at an enhanced rate. This study thus describes, to our knowledge, the first functional regulatory effect of tyrosine phosphorylation on a non-capsule-related protein in the pneumococcus, and suggests a link between the regulation of LytA-dependent autolysis of the cell and the biosynthesis of capsular polysaccharide.

Dual inhibition of DNA polymerase PolC and protein tyrosine phosphatase CpsB uncovers a novel antibiotic target.

Increasing antibiotic resistance is making the identification of novel antimicrobial targets critical. Recently, we discovered an inhibitor of protein tyrosine phosphatase CpsB, fascioquinol E (FQE), which unexpectedly inhibited the growth of Gram-positive pathogens. CpsB is a member of the polymerase and histidinol phosphate phosphatase (PHP) domain family. Another member of this family found in a variety of Gram-positive pathogens is DNA polymerase PolC. We purified the PHP domain from PolC (PolC(PHP)), and showed that this competes away FQE inhibition of CpsB phosphatase activity. Furthermore, we showed that this domain hydrolyses the 5'-p-nitrophenyl ester of thymidine-5'-monophosphate (pNP-TMP), which has been used as a measure of exonuclease activity. Finally, we showed that FQE not only inhibits the phosphatase activity of CpsB, but also ability of PolC(PHP) to catalyse the hydrolysis of pNP-TMP. This suggests that PolC may be the essential target of FQE, and that the PHP domain may represent an as yet untapped target for the development of novel antibiotics.

Human neutrophils kill Streptococcus pneumoniae via serine proteases.

Neutrophils, or polymorphonuclear leukocytes, comprise a crucial component of innate immunity, controlling bacterial and fungal infection through a combination of both oxidative and nonoxidative mechanisms. Indeed, neutrophils are believed to play an important role in controlling infection caused by the major human pathogen Streptococcus pneumoniae. However, the method by which neutrophils kill the pneumococcus as well as other Gram-positive bacteria, is not fully understood. We investigated human neutrophil killing of the pneumococcus in a complement-dependent opsonophagocytic assay. In contrast to other Gram-positive organisms, inhibition of the NADPH oxidase did not affect killing of S. pneumoniae. Supernatant from degranulated neutrophils killed the pneumococcus, suggesting a role for granular products. When neutrophil granule proteases were inhibited with either a protease mixture, or specific serine protease inhibitors 4-(2-Aminoethyl)benzenesulfonylfluoride and diisopropylfluorophosphate, killing by neutrophils was inhibited in a manner that correlated with increased intracellular survival. All three compounds inhibited intracellular activity of the three major neutrophil serine proteases: elastase, cathepsin G, and proteinase 3. Additionally, purified elastase and cathepsin G were sufficient to kill S. pneumoniae in a serine protease dependent-manner in in vitro assays. Inhibition studies using specific inhibitors of these serine proteases suggested that while each serine protease is sufficient to kill the pneumococcus, none is essential. Our findings show that Gram-positive pathogens are killed by human neutrophils via different mechanisms involving serine proteases.

Three surface exoglycosidases from Streptococcus pneumoniae, NanA, BgaA, and StrH, promote resistance to opsonophagocytic killing by human neutrophils.

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen and a leading cause of inflammatory infections such as pneumonia and otitis media. An important mechanism for host defense against S. pneumoniae is opsonophagocytic killing by neutrophils. To persist in the human host, the pneumococcus has developed strategies to evade opsonization and subsequent neutrophil-mediated killing. Utilizing a genomic approach, we identified NanA, the major pneumococcal neuraminidase, as a factor important for resistance to opsonophagocytic killing in ex vivo killing assays using human neutrophils. The effect of NanA was shown using both type 4 (TIGR4) and type 6A clinical isolates. NanA promotes this resistance by acting in conjunction with two other surface-associated exoglycosidases, BgaA, a beta-galactosidase, and StrH, an N-acetylglucosaminidase. Experiments using human serum showed that these exoglycosidases reduced deposition of complement component C3 on the pneumococcal surface, providing a mechanism for this resistance. Additionally, we have shown that antibodies in human serum do not contribute to this phenotype. These results demonstrate that deglycosylation of a human serum glycoconjugate(s) by the combined effects of NanA, BgaA, and StrH, is important for resistance to complement deposition and subsequent phagocytic killing of S. pneumoniae.

Resistance to mucosal lysozyme compensates for the fitness deficit of peptidoglycan modifications by Streptococcus pneumoniae.

The abundance of lysozyme on mucosal surfaces suggests that successful colonizers must be able to evade its antimicrobial effects. Lysozyme has a muramidase activity that hydrolyzes bacterial peptidoglycan and a non-muramidase activity attributable to its function as a cationic antimicrobial peptide. Two enzymes (PgdA, a N-acetylglucosamine deacetylase, and Adr, an O-acetyl transferase) that modify different sites on the peptidoglycan of Streptococcus pneumoniae have been implicated in its resistance to lysozyme in vitro. Here we show that the antimicrobial effect of human lysozyme is due to its muramidase activity and that both peptidoglycan modifications are required for full resistance by pneumococci. To examine the contribution of lysozyme and peptidoglycan modifications during colonization of the upper respiratory tract, competition experiments were performed with wild-type and pgdAadr mutant pneumococci in lysozyme M-sufficient (LysM(+/+)) and -deficient (LysM(-/-)) mice. The wild-type strain out-competed the double mutant in LysM(+/+), but not LysM(-/-) mice, indicating the importance of resistance to the muramidase activity of lysozyme during mucosal colonization. In contrast, strains containing single mutations in either pgdA or adr prevailed over the wild-type strain in both LysM(+/+) and LysM(-/-) mice. Our findings demonstrate that individual peptidoglycan modifications diminish fitness during colonization. The competitive advantage of wild-type pneumococci in LysM(+/+) but not LysM(-/-) mice suggests that the combination of peptidoglycan modifications reduces overall fitness, but that this is outweighed by the benefits of resistance to the peptidoglycan degrading activity of lysozyme.

Role of Streptococcus pneumoniae OM001 operon in capsular polysaccharide production, virulence and survival in human saliva.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia in all ages worldwide, and with ever-increasing antibiotic resistance, the understanding of its pathogenesis and spread is as important as ever. Recently, we reported the presence of a Low Molecular Weight Tyrosine Phosphatase (LMWPTP) Spd1837 in the pneumococcus. This protein is encoded in an operon, OM001 with two other genes, with previous work implicating this operon as important for pneumococcal virulence. Thus, we set out to investigate the role of the individual genes in the operon during pneumococcal pathogenesis. As LMWPTPs play a major role in capsular polysaccharide (CPS) biosynthesis in many bacteria, we tested the effect of mutating spd1837 and its adjacent genes, spd1836 and spd1838 on CPS levels. Our results suggest that individual deletion of the genes, including the LMWPTP, did not modulate CPS levels, in multiple conditions, and in different strain backgrounds. Following in vivo studies, Spd1836 was identified as a novel virulence factor during pneumococcal invasive disease, in both the lungs and blood, with this protein alone responsible for the effects of operon's role in virulence. We also showed that a deletion in spd1836, spd1838 or the overall OM001 operon reduced survival in human saliva during the conditions that mimic transmission compared to the wildtype strain. With studies suggesting that survival in human saliva may be important for transmission, this study identifies Spd1836 and Spd1838 as transmission factors, potentially facilitating the spread of the pneumococcus from person to person. Overall, this study hopes to further our understanding of the bacterial transmission that precedes disease and outbreaks.

Specific growth conditions induce a Streptococcus pneumoniae non-mucoidal, small colony variant and determine the outcome of its co-culture with Haemophilus influenzae.

Haemophilus influenzae and Streptococcus pneumoniae are known aetiologic agents of chronic otitis media, frequently as a multispecies infection. In this study, we show that the outcome of H. influenzae/S. pneumoniae interactions is dependent on the nutrient source. In continuous culture containing chemically defined media with lactose, S. pneumoniae was non-viable in mono-culture, and in co-culture remained non-viable until 288 h. With glucose, S. pneumoniae became non-viable in mono-culture, but uniquely existed in 3 distinct states in co-culture: parental cells (until 24 h), a dormant state until 336 h and its re-emergence as a non-mucoidal, small colony variant (SCV). The S. pneumoniae SCV was stable and whole genome sequencing showed three major single nucleotide polymorphisms in the SCV cells-cap3A (capsule biosynthesis pathway), fpg (DNA glycosylase of the DNA repair mechanism) and glutamate-5-kinase. Previously, fpg mutants have shown increased mutator rates, permitting bacterial survival against host-generated stresses. Transcriptomics showed these SCV cells up-regulated sugar transporters and toxin/antitoxin systems. An animal model revealed a reduced survival in the lungs and ear by SCV cells. This is the first study documenting the effect of carbon source and the development of a distinct S. pneumoniae cell type during H. influenzae/S. pneumoniae interactions.

Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents.

CpsB is a metal ion-dependent hydrolase involved in the biosynthesis of capsular polysaccharides in bacterial organisms. The enzyme has been proposed as a promising target for novel chemotherapeutics to combat antibiotic resistance. The crystal structure of CpsB indicated the presence of as many as three closely spaced metal ions, modelled as Mn2+, in the active site. While the preferred metal ion composition in vivo is obscure Mn2+ and Co2+ have been demonstrated to be most effective in reconstituting activity. Using isothermal titration calorimetry (ITC) we have demonstrated that, in contrast to the crystal structure, only two Mn2+ or Co2+ ions bind to a monomer of CpsB. This observation is in agreement with magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) data that indicate the presence of two weakly ferromagnetically coupled Co2+ ions in the active site of catalytically active CpsB. While CpsB is known to be a phosphoesterase we have also been able to demonstrate that this enzyme is efficient in hydrolyzing the ß-lactam substrate nitrocefin. Steady-state and stopped-flow kinetics measurements further indicated that phosphoesters and nitrocefin undergo catalysis in a conserved manner with a metal ion-bridging hydroxide acting as a nucleophile. Thus, the combined physicochemical studies demonstrate that CpsB is a novel member of the dinuclear metallohydrolase family.

The role of bacterial protein tyrosine phosphatases in the regulation of the biosynthesis of secreted polysaccharides.

SIGNIFICANCE: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria. RECENT ADVANCES: Deletion and overexpression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases have suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. CRITICAL ISSUES: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. FUTURE DIRECTIONS: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel antimicrobials. This is particularly the case for the polymerase and histidinol phosphatase family, which is predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism, and critical importance of these phosphatases in a wide range of bacteria.

Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production.

Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both gram-negative and -positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae, which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzy-dependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.

Neutrophil-toxin interactions promote antigen delivery and mucosal clearance of Streptococcus pneumoniae.

Delivery of Ag to inductive sites, such as nasal-associated lymphoid tissue (NALT) or GALT, is thought to promote mucosal immunity. Host and microbial factors that contribute to this process were investigated during model murine airway colonization by the pathogen Streptococcus pneumoniae. Colonization led to the deposition of released bacterial capsular Ag in the NALT in a manner consistent with trafficking through M cells. This Ag was derived from processing of bacteria in the lumen of the paranasal spaces rather than through invasion or sampling of intact bacteria. Neutrophils, which are recruited to the paranasal spaces where they associate with and may degrade bacteria, were required for efficient Ag delivery. Maximal Ag delivery to the NALT also required expression of the bacterial toxin pneumolysin. Pneumolysin and pneumolysin-expressing bacteria lysed neutrophils through pore formation in vitro. Accordingly, a pneumolysin-dependent loss of neutrophils, which correlated with the increased release of bacterial products, was observed in vivo. Thus, delivery of Ag to the NALT was enhanced by neutrophil-mediated generation of bacterial products together with bacterial-induced lysis of neutrophils. The impaired Ag delivery of pneumolysin-deficient bacteria was associated with diminished clearance from the mucosal surface. This study demonstrates how microbial-host interactions affect Ag delivery and the effectiveness of mucosal immunity.

The pneumococcal two-component signal transduction system RR/HK06 regulates CbpA and PspA by two distinct mechanisms.

We have previously shown that CbpA, a major pneumococcal virulence factor, is regulated by the two-component signal transduction system RR/HK06 (A. J. Standish, U. H. Stroeher, and J. C. Paton, Proc. Natl. Acad. Sci. USA 102:7701-7706, 2005). However, additional unidentified regulated factors appeared to be responsible for differences in adherence and the ability of Streptococcus pneumoniae to cause disease in a mouse model. Here, we identified a number of other regulated genes by overexpressing the system. cbpA, along with a cotranscribed upstream gene, showed substantial increases in expression when RR06 was overexpressed in S. pneumoniae strains D39 and TIGR4. However, there were no other similarities between these strains. In D39, rr06 overexpression decreased expression of numerous factors, including the major virulence factor gene pspA. Further investigation of cbpA regulation by RR/HK06, using mutants with mutations in both HK06 and RR06, suggested that rather than the norm, cbpA transcription was activated when RR06 was in the nonphosphorylated form. Although other factors, such as pspA and gls24, are regulated by this system, these genes appear to be repressed when RR06 is in its phosphorylated form.

Albomycin is an effective antibiotic, as exemplified with Yersinia enterocolitica and Streptococcus pneumoniae.

Albomycin belongs to the class of sideromycins, compounds composed of iron carriers linked to antibiotic moieties. Albomycin was found to be active against bacteria that have a functional ferric hydroxamate transport system meaning that bacteria will actively transport albomycin until they die. We examined the activity spectrum of albomycin for bacterial pathogens and found that Enterobacteriaceae except species of Proteus and Morganella were sensitive. Resistance in the two genera was due to the lack of the ferric hydroxamate transport system. Among Gram-positive bacteria, Staphylococcus aureus and Streptococcus pneumoniae were highly sensitive, whereas Streptococcus agalactiae, Streptococcus pyogenes, and Staphylococcus epidermidis were resistant. The in vivo efficacy of albomycin was examined in mice infected with S. pneumoniae or Yersinia enterocolitica. A single dose of 10mg albomycin/kg body weight reduced the colony-forming units of Y. enterocolitica by three to four orders of magnitude. A single dose of 1mg albomycin/kg body weight was sufficient to clear S. pneumoniae infections in mice. In direct competition experiments with wild-type S. pneumoniae and its albomycin-resistant mutant, the recovery rate of the mutant was lower than for the wild-type indicating that the mutant had reduced fitness in the mouse model. We conclude that albomycin is effective in clearing infections caused by both Gram-positive and Gram-negative bacteria in a mouse model. Albomycin treatment reduces the bacterial load allowing the immune system to remove residual albomycin-resistant bacteria, and as such would make albomycin-based antibiotics an adjunct to treatment. The ferrichrome transport system serves as a Trojan horse to get albomycin into bacteria.

The two-component signal transduction system RR06/HK06 regulates expression of cbpA in Streptococcus pneumoniae.

Streptococcus pneumoniae encounters a number of environmental niches in the body, including the nasopharynx, lungs, blood, middle ear, and brain. Recent studies have identified 13 putative two-component signal-transduction systems in S. pneumoniae, which are likely to be important for gene regulation in response to external stimuli. Here, we present conclusive evidence for the regulation of choline binding protein A (CbpA), a major pneumococcal virulence factor and protective antigen, by one of these two-component signal-transduction systems. We have demonstrated divergent expression of cbpA in unmarked hk06 and rr06 deletion mutants relative to wild-type S. pneumoniae D39 by using Western immunoblotting and real-time RT-PCR. Electrophoretic mobility-shift and solid-phase binding assays have demonstrated the binding of RR06 to the promoter region of cbpA, suggesting that RR06/HK06 directly regulates cbpA transcription. We have also shown that this system is important for the ability of the pneumococcus to adhere to epithelial cells in vitro and to survive and proliferate in an in vivo mouse model. Thus, the RR06/HK06 system has a significant role in pathogenesis, both in colonization and invasive disease.