Robert C. Holder

Characterization of the N-acetyl-α-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis .

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-d-glucosaminyl l-malate (GlcN-malate) from UDP-GlcNAc and l-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-d-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.

Dispersal of Group A streptococcal biofilms by the cysteine protease SpeB leads to increased disease severity in a murine model.

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980s there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.

One third of middle ear effusions from children undergoing tympanostomy tube placement had multiple bacterial pathogens

BackgroundBecause previous studies have indicated that otitis media may be a polymicrobial disease, we prospectively analyzed middle ear effusions of children undergoing tympanostomy tube placement with multiplex polymerase chain reaction for four otopathogens.MethodsMiddle ear effusions from 207 children undergoing routine tympanostomy tube placement were collected and were classified by the surgeon as acute otitis media (AOM) for purulent effusions and as otitis media with effusion (OME) for non-purulent effusions. DNA was isolated from these samples and analyzed with multiplex polymerase chain reaction for Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis.Results119 (57%) of 207 patients were PCR positive for at least one of these four organisms. 36 (30%) of the positive samples indicated the presence of more than one bacterial species. Patient samples were further separated into 2 groups based on clinical presentation at the time of surgery. Samples were categorized as acute otitis media (AOM) if pus was observed behind the tympanic membrane. If no pus was present, samples were categorized as otitis media with effusion (OME). Bacteria were identified in most of the children with AOM (87%) and half the children with OME (51%, p < 0.001). A single bacterial organism was detected in middle ear effusions from children with AOM more often than those with OME (74% versus 33%, p < 0.001). Haemophilus influenzae was the predominant single organism and caused 58% of all AOM in this study. Alloiococcus otitidis and Moraxella catarrhalis were more frequently identified in middle ear effusions than Streptococcus pneumoniae.ConclusionsHaemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis were identified in the middle ear effusions of some patients with otitis media. Overall, we found AOM is predominantly a single organism infection and most commonly from Haemophilus influenzae. In contrast, OME infections had a more equal distribution of single organisms, polymicrobial entities, and non-bacterial agents.

Loss of the Group A Streptococcus Regulator Srv Decreases Biofilm Formation In Vivo in an Otitis Media Model of Infection

ABSTRACT Group A Streptococcus (GAS) is a common causative agent of pharyngitis, but the role of GAS in otitis media is underappreciated. In this study, we sought to test the hypothesis that GAS colonizes the middle ear and establishes itself in localized, three-dimensional communities representative of biofilms. To test this hypothesis, the middle ears of chinchillas were infected with either a strain of GAS capable of forming biofilms in vitro (MGAS5005) or a strain deficient in biofilm formation due to the lack of the transcriptional regulator Srv (MGAS5005 Δsrv). Infection resulted in the formation of large, macroscopic structures within the middle ears of MGAS5005- and MGAS5005 Δsrv-infected animals. Plate counts, scanning electron microscopy, LIVE/DEAD staining, and Gram staining revealed a difference in the distributions of MGAS5005 versus MGAS5005 Δsrv in the infected samples. High numbers of CFU of MGAS5005 Δsrv were isolated from the middle ear effusion, and MGAS5005 Δsrv was found randomly distributed throughout the excised macroscopic structure. In contrast, MGAS5005 was found in densely packed microcolonies indicative of biofilms within the excised material from the middle ear. CFU levels of MGAS5005 from the effusion were significantly lower than that of MGAS5005 Δsrv early during the course of infection. Allelic replacement of the chromosomally encoded streptococcal cysteine protease (speB) in the MGAS5005 Δsrv background restored biofilm formation in vivo. Interestingly, our results suggest that GAS naturally forms a biofilm during otitis media but that biofilm formation is not required to establish infection following transbullar inoculation of chinchillas.

Srv mediated dispersal of streptococcal biofilms through SpeB is observed in CovRS+ strains.

Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds.

Point mutations within the streptococcal regulator of virulence (Srv) alter protein-DNA interactions and Srv function.

Group A Streptococcus (GAS) possesses a complex regulatory system enabling the organism to colonize a range of physiologically distinct host sites. Within this network of regulators is the streptococcal regulator of virulence (Srv). Srv is a member of the CRP/FNR family of transcriptional regulators and is most similar to pleiotropic regulatory factor A (PrfA), a positive regulator of virulence in Listeria monocytogenes. Members of this family possess a characteristic C-terminal helix-turn-helix motif (HTH) that facilitates binding to DNA targets. Genome scanning identified four targets in GAS that were similar to the consensus DNA target recognized by PrfA. Furthermore, previous amino acid sequence alignments identified conserved residues within the Srv HTH which are necessary for function in PrfA and CRP. Here we investigated the ability of Srv to interact with DNA and evaluated the role of the HTH in this interaction. Purified recombinant Srv (rSrv) was found to co-purify with an untagged form of Srv. Glutaraldehyde cross-linking and gel-filtration chromatography indicated that this co-purification is likely due to the ability of Srv to oligomerize. Electrophoretic mobility shift assays (EMSAs) demonstrated that rSrv retarded the mobility of DNA targets and a supershift analysis confirmed the observation was rSrv-dependent. Competition EMSA indicated that rSrv had a higher relative affinity for the DNA targets studied than non-specific DNA. Site-directed mutagenesis of residues predicted to be in or near the HTH resulted in a decrease or abrogation of DNA binding. Complementation of MGAS5005Deltasrv with one of these site-directed mutants failed to restore wild-type SpeB activity. Taken together, these data suggest that the Srv HTH is necessary for DNA binding and Srv function.

Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation

BackgroundGroup A Streptococcus (GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the srv mutant (MGAS5005Δsrv) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the srv mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δsrv to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δsrv was due only to the constitutive production of SpeB or to other changes inherent in the srv mutant strain. To address this question, we constructed a Δsrv ΔspeB double mutant through allelic replacement (MGAS5005Δsrv ΔspeB) and tested its ability to form biofilms in vitro.FindingsAllelic replacement of speB in the srv mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.ConclusionsThe constitutive production of SpeB by the srv mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.

Copper Tolerance and Characterization of a Copper-Responsive Operon, copYAZ, in an M1T1 Clinical Strain of Streptococcus pyogenes

UNLABELLED Infection with Streptococcus pyogenes is associated with a breadth of clinical manifestations ranging from mild pharyngitis to severe necrotizing fasciitis. Elevated levels of intracellular copper are highly toxic to this bacterium, and thus, the microbe must tightly regulate the level of this metal ion by one or more mechanisms, which have, to date, not been clearly defined. In this study, we have identified two virulence mechanisms by which S. pyogenes protects itself against copper toxicity. We defined a set of putative genes, copY (for a regulator), copA (for a P1-type ATPase), and copZ (for a copper chaperone), whose expression is regulated by copper. Our results indicate that these genes are highly conserved among a range of clinical S. pyogenes isolates. The copY, copA, and copZ genes are induced by copper and are transcribed as a single unit. Heterologous expression assays revealed that S. pyogenes CopA can confer copper tolerance in a copper-sensitive Escherichia coli mutant by preventing the accumulation of toxic levels of copper, a finding that is consistent with a role for CopA in copper export. Evaluation of the effect of copper stress on S. pyogenes in a planktonic or biofilm state revealed that biofilms may aid in protection during initial exposure to copper. However, copper stress appears to prevent the shift from the planktonic to the biofilm state. Therefore, our results indicate that S. pyogenes may use several virulence mechanisms, including altered gene expression and a transition to and from planktonic and biofilm states, to promote survival during copper stress. IMPORTANCE Bacterial pathogens encounter multiple stressors at the host-pathogen interface. This study evaluates a virulence mechanism(s) utilized by S. pyogenes to combat copper at sites of infection. A better understanding of pathogen tolerance to stressors such as copper is necessary to determine how host-pathogen interactions impact bacterial survival during infections. These insights may lead to the identification of novel therapeutic targets that can be used to address antibiotic resistance.

Otopathogens Detected in Middle Ear Fluid Obtained during Tympanostomy Tube Insertion: Contrasting Purulent and Non-Purulent Effusions.

Otitis media is a prominent disease among children. Previous literature indicates that otitis media is a polymicrobial disease, with Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis and Moraxella catarrhalis being the most commonly associated bacterial pathogens. Recent literature suggests that introduction of pneumococcal conjugate vaccines has had an effect on the etiology of otitis media. Using a multiplex PCR procedure, we sought to investigate the presence of the aforementioned bacterial pathogens in middle ear fluid collected from children undergoing routine tympanostomy tube placement at Wake Forest Baptist Medical Center during the period between January 2011 and March 2014. In purulent effusions, one or more bacterial organisms were detected in ~90% of samples. Most often the presence of H. influenzae alone was detected in purulent effusions (32%; 10 of 31). In non-purulent effusions, the most prevalent organism detected was A. otitidis (26%; 63 of 245). Half of the non-purulent effusions had none of these otopathogens detected. In purulent and non-purulent effusions, the overall presence of S. pneumoniae was lower (19%; 6 of 31, and 4%; 9 of 245, respectively) than that of the other pathogens being identified. The ratio of the percentage of each otopathogen identified in purulent vs. non-purulent effusions was >1 for the classic otopathogens but not for A. otitidis.

Activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (PIV5) P/V mutant

Macrophages regulate immune responses during many viral infections, and can be a major determinant of pathogenesis, virus replication and immune response to infection. Here, we have addressed the question of the outcome of infection of primary human macrophages with parainfluenza virus 5 (PIV5) and a PIV5 mutant (P/V-CPI-) that is unable to counteract interferon (IFN) responses. In cultures of naïve monocyte-derived macrophages (MDMs), WT PIV5 established a highly productive infection, whereas the P/V-CPI- mutant was restricted for replication in MDMs by IFN-beta. Restricted replication in vitro was relieved in MDM that had been activated by prior exposure to heat killed Gram positive bacteria, including Listeria monocytogenes, Streptococcus pyogenes, and Bacillus anthracis. Enhanced replication of the P/V mutant in MDM previously activated by bacterial components correlated with a reduced ability to produce IFN-beta in response to virus infection, whereas IFN signaling was intact. Activated MDM were found to upregulate the synthesis of IRAK-M, which has been previously shown to negatively regulate factors involved in TLR signaling and IFN-beta production. We discuss these results in terms of the implications for mixed bacteria-virus infections and for the use of live RNA virus vectors that have been engineered to be attenuated for IFN sensitivity.