Staphylococcus aureus Hyaluronidase Is a CodY-Regulated Virulence Factor

ABSTRACTStaphylococcus aureusis a Gram-positive pathogen that causes a diverse range of bacterial infections. InvasiveS. aureusstrains secrete an extensive arsenal of hemolysins, immunomodulators, and exoenzymes to cause disease. Our studies have focused on the secreted enzyme hyaluronidase (HysA), which cleaves the hyaluronic acid polymer at the β-1,4 glycosidic bond. In the study described in this report, we have investigated the regulation and contribution of this enzyme toS. aureuspathogenesis. Using the Nebraska Transposon Mutant Library (NTML), we identified eight insertions that modulate extracellular levels of HysA activity. Insertions in thesigBoperon, as well as in genes encoding the global regulators SarA and CodY, significantly increased HysA protein levels and activity. By altering the availability of branched-chain amino acids, we further demonstrated CodY-dependent repression of HysA activity. Additionally, through mutation of the CodY binding box upstream ofhysA, the repression of HysA production was lost, suggesting that CodY is a direct repressor ofhysAexpression. To determine whether HysA is a virulence factor, a ΔhysAmutant of a community-associated methicillin-resistantS. aureus(CA-MRSA) USA300 strain was constructed and found to be attenuated in a neutropenic, murine model of pulmonary infection. Mice infected with this mutant strain exhibited a 4-log-unit reduction in bacterial burden in their lungs, as well as reduced lung pathology and increased levels of pulmonary hyaluronic acid, compared to mice infected with the wild-type, parent strain. Taken together, these results indicate thatS. aureushyaluronidase is a CodY-regulated virulence factor.

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sae is essential for expression of the staphylococcal adhesins Eap and Emp

Microbiology ◽

10.1099/mic.0.27902-0 ◽

2005 ◽

Vol 151 (6) ◽

pp. 1789-1800 ◽

Cited By ~ 56

Author(s):

Niamh Harraghy ◽

Jan Kormanec ◽

Christiane Wolz ◽

Dagmar Homerova ◽

Christiane Goerke ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Extracellular Matrix ◽

Virulence Factor ◽

Eukaryotic Cells ◽

Transcription Start Point ◽

Chronic Infections ◽

Transcription Start ◽

Regulatory Mutants ◽

Global Regulators ◽

Glucose Analysis

Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae.

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Bacteriocins of Non-aureus Staphylococci Isolated from Bovine Milk

Applied and Environmental Microbiology ◽

10.1128/aem.01015-17 ◽

2017 ◽

Vol 83 (17) ◽

Cited By ~ 24

Author(s):

Domonique A. Carson ◽

Herman W. Barkema ◽

Sohail Naushad ◽

Jeroen De Buck

Keyword(s):

Staphylococcus Aureus ◽

Bacterial Infections ◽

Genome Mining ◽

Bovine Milk ◽

Gene Clusters ◽

Antibiotic Use ◽

Content Type ◽

Treatment And Prevention ◽

Whole Genomes

ABSTRACT Non-aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine udder, potentially protect the udder against infection by major mastitis pathogens due to bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bovine Staphylococcus aureus. Furthermore, inhibiting isolates were tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. We determined the presence of bacteriocin clusters in NAS whole genomes using genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and noninhibiting isolates and determined the genetic organization of any identified bacteriocin biosynthetic gene clusters. Forty isolates from 9 species (S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus) inhibited growth of S. aureus in vitro, 23 isolates of which, from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus, also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. A total of 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications. IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus) costs Canadian dairy producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With increasing antibiotic resistance and regulations regarding use, there is impetus to explore bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of bacterial infections. We examined the ability of 441 NAS bacteria from Canadian bovine milk samples to inhibit growth of S. aureus in the laboratory. Overall, 9% inhibited growth of S. aureus and 58% of those also inhibited MRSA. In NAS whole-genome sequences, we identified >21% of NAS as having bacteriocin genes. Our study represents a foundation to further explore NAS bacteriocins for clinical use.

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Transcriptional Profiling Analysis of the Global Regulator NorG, a GntR-Like Protein of Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.05847-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6207-6214 ◽

Cited By ~ 18

Author(s):

Q. C. Truong-Bolduc ◽

P. M. Dunman ◽

T. Eidem ◽

D. C. Hooper

Keyword(s):

Staphylococcus Aureus ◽

Target Genes ◽

Efflux Pump ◽

Transcriptional Profiling ◽

Fold Increase ◽

Regulatory Function ◽

Drug Transporter ◽

Content Type ◽

Global Regulators ◽

Inorganic Ion

The GntR-like protein NorG has been shown to affectStaphylococcus aureusgenes involved in resistance to quinolones and β-lactams, such as those encoding the NorB and AbcA transporters. To identify the target genes regulated by NorG, we carried out transcriptional-profiling assays usingS. aureusRN6390 and its isogenicnorG::catmutant. Our data showed that NorG positively affected the transcription of global regulatorsmgrA,arlS, andsarZ. The three putative drug efflux pump genes most positively affected by NorG were the NorB efflux pump (5.1-fold), the MmpL-like protein SACOL2566 (5.2-fold), and the BcrA-like drug transporter SACOL2525 (5.7-fold) genes. TheS. aureuspredicted MmpL protein showed 53% homology with the MmpL lipid transporter ofMycobacterium tuberculosis, and the putative SACOL2525 protein showed 87% homology with the bacitracin drug transporter BcrA ofStaphylococcus hominis. Two pump genes most negatively affected by NorG were the NorC (4-fold) and AbcA (6-fold) genes. Other categories of genes, such as those participating in amino acid, inorganic ion, or nucleotide transporters and metabolism, were also affected by NorG. Real-time reverse transcription (RT)-PCR assays formgrA,arlS,sarZ,norB,norC,abcA,mmpL, andbcrA-like were carried out to verify microarray data and showed the same level of up- or downregulation by NorG. ThenorGmutant showed a 2-fold increase in resistance to norfloxacin and rhodamine, both substrates of the NorC transporter, which is consistent with the resistance phenotype conferred by overexpression ofnorCon a plasmid. These data indicate that NorG has broad regulatory function inS. aureus.

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RbsR Activates Capsule but Represses therbsUDKOperon in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00640-15 ◽

2015 ◽

Vol 197 (23) ◽

pp. 3666-3675 ◽

Cited By ~ 13

Author(s):

Mei G. Lei ◽

Chia Y. Lee

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factor ◽

Transcriptional Activation ◽

Promoter Region ◽

Mobility Shift ◽

Content Type ◽

Virulence Regulation ◽

Important Virulence Factor ◽

Dna Fragment ◽

Mobility Shift Assay

ABSTRACTStaphylococcus aureuscapsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of thecapoperon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of thecappromoter. To search for potential proteins which directly interact with thecappromoter region (Pcap), we directly analyzed the proteins interacting with the PcapDNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulatecapgene expression by specifically binding to thecappromoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed thatrbsRwas directly controlled by SigB and that RbsR was a repressor of therbsUDKoperon, involved in ribose uptake and phosphorylation. The repression ofrbsUDKby RbsR could be derepressed byd-ribose. However,d-ribose did not affect RbsR activation of capsule.IMPORTANCEStaphylococcus aureusis an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression ofrbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits therbsoperon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence inS. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

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In VitroStudies Indicate a High Resistance Potential for the Lantibiotic Nisin in Staphylococcus aureus and Define a Genetic Basis for Nisin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01077-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2362-2368 ◽

Cited By ~ 49

Author(s):

Katy L. Blake ◽

Chris P. Randall ◽

Alex J. O'Neill

Keyword(s):

Staphylococcus Aureus ◽

Bacterial Infections ◽

Fold Increase ◽

Peptide Antibiotics ◽

Uncharacterized Protein ◽

Content Type ◽

Development Of Resistance ◽

Genes Encoding ◽

Bacterial Fitness

ABSTRACTLantibiotics such as nisin (NIS) are peptide antibiotics that may have a role in the chemotherapy of bacterial infections. A perceived benefit of lantibiotics for clinical use is their low propensity to select resistance, although detailed resistance studies with relevant bacterial pathogens are lacking. Here we examined the development of resistance to NIS inStaphylococcus aureus, establishing that mutants, including small-colony variants, exhibiting substantial (4- to 32-fold) reductions in NIS susceptibility could be selected readily. Comparative genome sequencing of a single NISrmutant exhibiting a 32-fold increase in NIS MIC revealed the presence of only two mutations, leading to the substitutions V229G in the purine operon repressor, PurR, and A208E in an uncharacterized protein encoded by SAOUHSC_02955. Independently selected NISrmutants also harbored mutations in the genes encoding these products. Reintroduction of these mutations into theS. aureuschromosome alone and in combination revealed that SAOUHSC_02955(A208E) made the primary contribution to the resistance phenotype, conferring up to a 16-fold decrease in NIS susceptibility. Bioinformatic analyses suggested that this gene encodes a sensor histidine kinase, leading us to designate it “nisin susceptibility-associated sensor (nsaS).” Doubling-time determinations and mixed-culture competition assays between NISrand NISsstrains indicated that NIS resistance had little impact on bacterial fitness, and resistance was stable in the absence of selection. The apparent ease with whichS. aureuscan develop and maintain NIS resistancein vitrosuggests that resistance to NIS and other lantibiotics with similar modes of action would arise in the clinic if these agents are employed as chemotherapeutic drugs.

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Dietary Polyunsaturated Fatty Acids Increase Survival and Decrease Bacterial Load during Septic Staphylococcus aureus Infection and Improve Neutrophil Function in Mice

Infection and Immunity ◽

10.1128/iai.02349-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 514-521 ◽

Cited By ~ 21

Author(s):

Sara L. Svahn ◽

Louise Grahnemo ◽

Vilborg Pálsdóttir ◽

Intawat Nookaew ◽

Karl Wendt ◽

...

Keyword(s):

Fatty Acids ◽

Staphylococcus Aureus ◽

Dietary Fat ◽

High Fat Diet ◽

Bacterial Infections ◽

Saturated Fatty Acids ◽

Bacterial Load ◽

Neutrophil Function ◽

High Fat ◽

Content Type

Severe infection, including sepsis, is an increasing clinical problem that causes prolonged morbidity and substantial mortality. At present, antibiotics are essentially the only pharmacological treatment for sepsis. The incidence of resistance to antibiotics is increasing; therefore, it is critical to find new therapies for sepsis.Staphylococcus aureusis a major cause of septic mortality. Neutrophils play an important role in the defense against bacterial infections. We have shown that a diet with high levels of dietary saturated fatty acids decreases survival in septic mice, but the mechanisms behind this remain elusive. The aim of the present study was to investigate how the differences in dietary fat composition affect survival and bacterial load after experimental septic infection and neutrophil function in uninfected mice. We found that, afterS. aureusinfection, mice fed a polyunsaturated high-fat diet (HFD-P) for 8 weeks had increased survival and decreased bacterial load during sepsis compared with mice fed a saturated high-fat diet (HFD-S), similar to mice fed a low-fat diet (LFD). Uninfected mice fed HFD-P had a higher frequency of neutrophils in bone marrow than mice fed HFD-S. In addition, mice fed HFD-P had a higher frequency of neutrophils recruited to the site of inflammation in response to peritoneal injection of thioglycolate than mice fed HFD-S. Differences between the proportion of dietary protein and carbohydrate did not affect septic survival at all. In conclusion, polyunsaturated dietary fat increased both survival and efficiency of bacterial clearance during septicS. aureusinfection. Moreover, this diet increased the frequency and chemotaxis of neutrophils, key components of the immune response toS. aureusinfections.

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Staphylococcus aureus Protein A Promotes Immune Suppression

mBio ◽

10.1128/mbio.00764-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 34

Author(s):

Scott D. Kobayashi ◽

Frank R. DeLeo

Keyword(s):

Staphylococcus Aureus ◽

B Cell ◽

Immune Evasion ◽

Bacterial Infections ◽

Binding Capacity ◽

Protein A ◽

Strong Support ◽

The United States ◽

Content Type

ABSTRACTStaphylococcus aureusis a prominent cause of human infections worldwide and is notorious for its ability to acquire resistance to antibiotics. Methicillin-resistantS. aureus(MRSA), in particular, is endemic in hospitals and is the most frequent cause of community-associated bacterial infections in the United States. Inasmuch as treatment options for severe MRSA infections are limited, there is need for a vaccine that protects against such infections. However, recent efforts to generate a staphylococcal vaccine have met with little success in human clinical trials. These failures are somewhat puzzling, since the vaccine antigens tested promote opsonophagocytosisin vitroand confer protection in animal infection models. One possibility is that the pathogen inhibits (and/or fails to elicit) the development of protective immunity in humans. Indeed,S. aureusproduces numerous molecules that can potentially promote immune evasion, including protein A (SpA), an immunoglobulin (Ig)-binding protein present on the bacterial surface and freely secreted into the extracellular environment. SpA binds the Fc region of antibody and the Fab regions of the B-cell receptor, processes that are known to block opsonophagocytosis and cause B-cell deathin vitro. In a recent study, Falugi et al. [F. Falugi, H. K. Kim, D. M. Missiakas, and O. Schneewind, mBio 4(5):e00575-13, 2013] showed that vaccination withspamutantS. aureusstrains lacking antibody Fc- and/or Fab-binding capacity protects against subsequent challenge with the USA300 epidemic strain. The findings provide strong support for the idea that SpA promotesS. aureusimmune evasionin vivoand form the foundation for a new approach in our efforts to develop a vaccine that prevents severeS. aureusinfections.

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Targeting Alpha Toxin To Mitigate Its Lethal Toxicity in Ferret and Rabbit Models of Staphylococcus aureus Necrotizing Pneumonia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02456-16 ◽

2017 ◽

Vol 61 (4) ◽

Cited By ~ 20

Author(s):

Binh An Diep ◽

Jamese J. Hilliard ◽

Vien T. M. Le ◽

Christine Tkaczyk ◽

Hoan N. Le ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bacterial Infections ◽

Passive Immunization ◽

Adjunctive Treatment ◽

Alpha Toxin ◽

Necrotizing Pneumonia ◽

Promising Candidate ◽

Content Type ◽

Serious Bacterial Infections ◽

Mrsa Strains

ABSTRACT The role broad-spectrum antibiotics play in the spread of antimicrobial resistance, coupled with their effect on the healthy microbiome, has led to advances in pathogen-specific approaches for the prevention or treatment of serious bacterial infections. One approach in clinical testing is passive immunization with a monoclonal antibody (MAb) targeting alpha toxin for the prevention or treatment of Staphylococcus aureus pneumonia. Passive immunization with the human anti-alpha toxin MAb, MEDI4893*, has been shown to improve disease outcome in murine S. aureus pneumonia models. The species specificity of some S. aureus toxins necessitates testing anti-S. aureus therapeutics in alternate species. We developed a necrotizing pneumonia model in ferrets and utilized an existing rabbit pneumonia model to characterize MEDI4893* protective activity in species other than mice. MEDI4893* prophylaxis reduced disease severity in ferret and rabbit pneumonia models against both community-associated methicillin-resistant S. aureus (MRSA) and hospital-associated MRSA strains. In addition, adjunctive treatment of MEDI4893* with either vancomycin or linezolid provided enhanced protection in rabbits relative to the antibiotics alone. These results confirm that MEDI4893 is a promising candidate for immunotherapy against S. aureus pneumonia.

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Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00162-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 2929-2936 ◽

Cited By ~ 8

Author(s):

Thomas Lewandowski ◽

Jianzhong Huang ◽

Frank Fan ◽

Shannon Rogers ◽

Daniel Gentry ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factor ◽

Survival Rates ◽

Extracellular Proteins ◽

High Survival ◽

Invasive Infection ◽

Loss Of Function ◽

Content Type ◽

Factor Production ◽

Virulence Factor Production

ABSTRACTInhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors inStaphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated withS. aureuspathogenicity. Consequently,S. aureusFMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-typeS. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection.

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CodY Deletion EnhancesIn VivoVirulence of Community-Associated Methicillin-Resistant Staphylococcus aureus Clone USA300

Infection and Immunity ◽

10.1128/iai.06172-11 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2382-2389 ◽

Cited By ~ 36

Author(s):

Christopher P. Montgomery ◽

Susan Boyle-Vavra ◽

Agnès Roux ◽

Kazumi Ebine ◽

Abraham L. Sonenshein ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Genes ◽

Target Genes ◽

Gene Encoding ◽

Methicillin Resistant ◽

Content Type ◽

Global Regulators ◽

Alpha Hemolysin

ABSTRACTTheStaphylococcus aureusglobal regulator CodY responds to nutrient availability by controlling the expression of target genes.In vitro, CodY represses the transcription of virulence genes, but it is not known if CodY also represses virulencein vivo. The dominant community-associated methicillin-resistantS. aureus(CA-MRSA) clone, USA300, is hypervirulent and has increased transcription of global regulators and virulence genes; these features are reminiscent of a strain defective in CodY. Sequence analysis revealed, however, that thecodYgenes of USA300 and other sequencedS. aureusisolates are not significantly different from thecodYgenes in strains known to have active CodY.codYwas expressed in USA300, as well as in other pulsotypes assessed. Deletion ofcodYfrom a USA300 clinical isolate resulted in modestly increased expression of the global regulatorsagrandsaeRS, as well as the gene encoding the toxin alpha-hemolysin (hla). A substantial increase (>30-fold) in expression of thelukF-PVgene, encoding part of the Panton-Valentine leukocidin (PVL), was observed in thecodYmutant. All of these expression differences were reversed by complementation with a functionalcodYgene. Moreover, purified CodY protein bound upstream of thelukSF-PVoperon, indicating that CodY directly represses expression oflukSF-PV. Deletion ofcodYincreased the virulence of USA300 in necrotizing pneumonia and skin infection. Interestingly, deletion oflukSF-PVfrom thecodYmutant did not attenuate virulence, indicating that the hypervirulence of thecodYmutant was not explained by overexpression of PVL. These results demonstrate that CodY is active in USA300 and that CodY-mediated repression restrains the virulence of USA300.

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