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.Alistair J. Standish, Angela A. Salim, Robert J. Capon, Renato Moron

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

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 Wzydependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.Alistair J. Standish, Angela A. Salim, Hua Zhang, Robert J. Capon and Renato Moron

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

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 β-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

A Non-local Source of Irish Chalcolithic and Early Bronze Age Gold

Lead isotope analyses of 50 Irish Chalcolithic and Early Bronze Age gold artefacts favour a gold source in southern Ireland. However when combined with major element analysis, the artefacts are not consistent with any Irish gold deposit analysed to date. Understanding the lead isotope signatures of ore deposits within a study region allows informed inferences to be drawn regarding the likelihood that an unanalysed ore deposit was exploited in the past. If an Irish gold source is assumed, then the gold is most likely to have originated from deposits hosted by Old Red Sandstone in the Variscan ore field of south-west Ireland. However, based on our current understanding of mineralisation in the region, this scenario is considered unlikely. A non-Irish source for the gold is therefore preferred – a scenario that may favour cosmologically-driven acquisition, ie, the deliberate procurement of a material from distant or esoteric sources. Available geochemical data, combined with current archaeological evidence, favour the alluvial deposits of south-west Britain as the most likely source of the gold.</jats:p

Effect of peptidoglycan modifying enzymes PgdA and Adr on relative fitness during murine colonization in the presence and absence of lysozyme M.

<p>LysM^+/+ and LysM^−/− mice were challenged with equal inocula of the wild-type (WT) strain or revertant and the defined mutant indicated, and the density of each strain was determined in upper respiratory tract lavages 3 days post-inoculation. Each symbol represents the competitive index value for an individual animal. The competitive index was calculated based on the ratio of mutant to WT bacteria in nasal lavages compared to the ratio of mutant to WT bacteria in the inoculum. The dotted line is at a value of one; a value greater than one indicates the mutant out-competes the WT, a value less than or equal to one indicates the WT out-competes the mutant. A) <i>pgdAadr</i> vs. WT (^*** p = 0.001). B) <i>pgdAadr</i> vs. the revertant (<i>pgdA+ adr+</i>) (^** p = 0.004). C) <i>pgdA</i> vs. WT. D) <i>adr</i> vs. WT.</p

Effect of peptidoglycan modifying enzymes PgdA and Adr on hydrolysis of pneumococcal cell walls in the presence of lysozyme.

<p>Hydrolysis of peptidoglycan (50 µg/ml), purified from the wild-type (WT) strain or the defined mutants indicated with lysozyme (100 µg/ml) from A) chicken egg (+L) or B) human (+Hu L). Representative experiment showing percentage of hydrolysis based on the optical density (OD 600 nm) of each reaction at time 0 min.</p

Effect of peptidoglycan modifying enzymes PgdA and Adr on viability of pneumococci in the presence of lysozyme.

<p>Once the broth culture of the wild-type (WT) strain or the defined mutants indicated reached mid-log phase, lysozyme (100 µg/ml) was added and viable counts (CFU/ml) were measured 5 hrs later. Strains tested were in a <i>lytA</i> background to eliminate effects of autolysis. Conditions included A) chicken egg lysozyme (+L) and B) recombinant human lysozyme (+Hu L) or heat inactivated recombinant human lysozyme (+Hu IL). Graphs are based on four independent determinations ±S.D. (^* p<0.05).</p