Tools, Strains, and Strategies To Effectively Conduct Anaerobic and Aerobic Transcriptional Reporter Screens and Assays in Staphylococcus aureus

Staphylococcus aureus is a human pathogen and a leading cause of infectious disease-related illness and death worldwide. For S. aureus to successfully colonize and invade host tissues, it must tightly control the expression of genes encoding virulence factors.

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Relationship between Vancomycin MIC and Virulence Gene Expression in Clonal Complexes of Methicillin-Susceptible Staphylococcus aureus Strains Isolated from Left-Sided Endocarditis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01579-19 ◽

2020 ◽

Vol 64 (3) ◽

Author(s):

Juan M. Pericàs ◽

Carlos Cervera ◽

Cristina Garcia-de-la-Mària ◽

Batu K. Sharma-Kuinkel ◽

Rachelle Gonzales ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Virulence Genes ◽

Multivariable Analysis ◽

Virulence Gene ◽

Mortality Rates ◽

Content Type ◽

Virulence Gene Expression ◽

Vancomycin Mics ◽

Genes Encoding

ABSTRACT Higher vancomycin MICs have been associated with more complicated courses and higher mortality rates in patients with Staphylococcus aureus bacteremia and infective endocarditis (IE). The aim of this study was to investigate whether the strains belonging to the cohort of 93 patients from a previously published study in which patients with strains with vancomycin MICs of ≥1.5 μg/ml presented higher mortality rates and systemic emboli than patients with strains with vancomycin MICs of <1.5 μg/ml had specific patterns of virulence factors, clonal complex (CC) types, or the ability to form biofilms. Vancomycin MICs were determined by Etest, and the isolates underwent spa typing to infer the CC, biofilm studies, a thrombin-induced platelet microbicidal assay, and multiplex PCR for the presence of virulence genes. We found no differences in genes encoding adhesins, toxins, or other putative virulence genes according to the vancomycin MIC group. CC30, CC34, and CC45 represented nearly half of the isolates, and there was no association with the vancomycin MIC. agr subgroups I and III predominated, with no association with the vancomycin MIC. Isolates with higher vancomycin MICs exhibited a poorer ability to form biofilms with and without the presence of vancomycin (2.03 versus 2.48 [P < 0.001], respectively, for isolates with higher vancomycin MICs and 2.60 versus 2.87 [P = 0.022], respectively, for isolates with lower vancomycin MICs). In the multivariable analysis, efb and V8 were risk factors for major emboli (adjusted odds ratio [aOR] = 7.5 and 95% confidence interval [CI] = 1.2 to 46.6 for efb, and aOR = 3.9 and 95% CI = 1.1 to 14.1 for V8), whereas no genotypic predictors of in-hospital mortality were found. No clear associations between genes encoding virulence factors, agr type, clonal complexes, mortality, and major embolic events according to vancomycin MIC group were found.

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Phosphorylation of MgrA and Its Effect on Expression of the NorA and NorB Efflux Pumps of Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00018-10 ◽

2010 ◽

Vol 192 (10) ◽

pp. 2525-2534 ◽

Cited By ~ 55

Author(s):

Que Chi Truong-Bolduc ◽

David C. Hooper

Keyword(s):

Staphylococcus Aureus ◽

Multidrug Resistance ◽

Virulence Factors ◽

Double Mutant ◽

Efflux Pumps ◽

Efflux Transporters ◽

Global Regulator ◽

Binding Assays ◽

Genes Encoding ◽

Gel Shift

ABSTRACT MgrA is a global regulator in Staphylococcus aureus that controls the expression of diverse genes encoding virulence factors and multidrug resistance (MDR) efflux transporters. We identified pknB, which encodes the (Ser/Thr) kinase PknB, in the S. aureus genome. PknB was able to autophosphorylate as well as phosphorylate purified MgrA. We demonstrated that rsbU, which encodes a Ser/Thr phosphatase and is involved in the activation of the SigB regulon, was able to dephosphorylate MgrA-P but not PknB-P. Serines 110 and 113 of MgrA were found to be phosphorylated, and Ala substitutions at these positions resulted in reductions in the level of phosphorylation of MgrA. DNA gel shift binding assays using norA and norB promoters showed that MgrA-P was able to bind the norB promoter but not the norA promoter, a pattern which was the reverse of that for unphosphorylated MgrA. The double mutant MgrAS110A-S113A bound to the norA promoter but not the norB promoter. The double mutant led to a 2-fold decrease in norA transcripts and a 2-fold decrease in the MICs of norfloxacin and ciprofloxacin in strain RN6390. Thus, phosphorylation of MgrA results in loss of binding to the norA promoter, but with a gain of the ability to bind the norB promoter. Loss of the ability to phosphorylate MgrA by Ala substitution resulted in increased repression of norA expression and in reductions in susceptibilities to NorA substrates.

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Variable Expressions of Staphylococcus aureus Bicomponent Leucotoxins Semiquantified by Competitive Reverse Transcription-PCR

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.3931-3938.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 3931-3938 ◽

Cited By ~ 32

Author(s):

StÃ¯Â¿Â½phane Bronner ◽

Patricia Stoessel ◽

Alain Gravet ◽

Henri Monteil ◽

Gilles PrÃ¯Â¿Â½vost

Keyword(s):

Staphylococcus Aureus ◽

Reverse Transcription ◽

Regulatory System ◽

Optimization Procedure ◽

Reverse Transcription Pcr ◽

Expression Of Genes ◽

Genes Encoding ◽

Specific Mrnas ◽

Casamino Acids ◽

Global Regulatory System

ABSTRACT A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/104 CFU to 102 mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system inS. aureus, except that expression of hlgA was not affected in the agr mutant.

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Finding of Agr Phase Variants in Staphylococcus aureus

mBio ◽

10.1128/mbio.00796-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 3

Author(s):

Vishal Gor ◽

Aya J. Takemura ◽

Masami Nishitani ◽

Masato Higashide ◽

Veronica Medrano Romero ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Phase Variation ◽

Accessory Gene ◽

Content Type ◽

Accessory Gene Regulator ◽

Immune Mediated ◽

A Minor ◽

First Time ◽

Phase Variants

ABSTRACT Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. IMPORTANCE Staphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

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Derivatives of Plant Phenolic Compound Affect the Type III Secretion System of Pseudomonas aeruginosa via a GacS-GacA Two-Component Signal Transduction System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00732-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 36-43 ◽

Cited By ~ 47

Author(s):

Akihiro Yamazaki ◽

Jin Li ◽

Quan Zeng ◽

Devanshi Khokhani ◽

William C. Hutchins ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Phenolic Compounds ◽

Virulence Factors ◽

Type Iii Secretion ◽

Small Rnas ◽

Type Iii Secretion System ◽

Secretion System ◽

Human Pathogen ◽

Content Type ◽

Type Iii

ABSTRACTAntibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability.Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening ofexoS, which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers ofP. aeruginosaPAO1. These compounds alterexoStranscription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression ofexoSthrough the GacSA-RsmYZ-RsmA-ExsA regulatory pathway.

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The Streptococcal Protease SpeB Antagonizes the Biofilms of the Human Pathogen Staphylococcus aureus USA300 through Cleavage of the Staphylococcal SdrC Protein

Journal of Bacteriology ◽

10.1128/jb.00008-20 ◽

2020 ◽

Vol 202 (11) ◽

Author(s):

Katelyn E. Carothers ◽

Zhong Liang ◽

Jeffrey Mayfield ◽

Deborah L. Donahue ◽

Mijoon Lee ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Proteolytic Activity ◽

Streptococcus Pyogenes ◽

Human Pathogen ◽

Content Type ◽

Functional Studies ◽

Human Proteins ◽

Group A ◽

Biofilm Disruption

ABSTRACT Streptococcus pyogenes, or group A Streptococcus (GAS), is both a pathogen and an asymptomatic colonizer of human hosts and produces a large number of surface-expressed and secreted factors that contribute to a variety of infection outcomes. The GAS-secreted cysteine protease SpeB has been well studied for its effects on the human host; however, despite its broad proteolytic activity, studies on how this factor is utilized in polymicrobial environments are lacking. Here, we utilized various forms of SpeB protease to evaluate its antimicrobial and antibiofilm properties against the clinically important human colonizer Staphylococcus aureus, which occupies niches similar to those of GAS. For our investigation, we used a skin-tropic GAS strain, AP53CovS+, and its isogenic ΔspeB mutant to compare the production and activity of native SpeB protease. We also generated active and inactive forms of recombinant purified SpeB for functional studies. We demonstrate that SpeB exhibits potent biofilm disruption activity at multiple stages of S. aureus biofilm formation. We hypothesized that the surface-expressed adhesin SdrC in S. aureus was cleaved by SpeB, which contributed to the observed biofilm disruption. Indeed, we found that SpeB cleaved recombinant SdrC in vitro and in the context of the full S. aureus biofilm. Our results suggest an understudied role for the broadly proteolytic SpeB as an important factor for GAS colonization and competition with other microorganisms in its niche. IMPORTANCE Streptococcus pyogenes (GAS) causes a range of diseases in humans, ranging from mild to severe, and produces many virulence factors in order to be a successful pathogen. One factor produced by many GAS strains is the protease SpeB, which has been studied for its ability to cleave and degrade human proteins, an important factor in GAS pathogenesis. An understudied aspect of SpeB is the manner in which its broad proteolytic activity affects other microorganisms that co-occupy niches similar to that of GAS. The significance of the research reported herein is the demonstration that SpeB can degrade the biofilms of the human pathogen Staphylococcus aureus, which has important implications for how SpeB may be utilized by GAS to successfully compete in a polymicrobial environment.

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The purine biosynthesis regulator PurR moonlights as a virulence regulator in Staphylococcus aureus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904280116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13563-13572 ◽

Cited By ~ 9

Author(s):

William E. Sause ◽

Divya Balasubramanian ◽

Irnov Irnov ◽

Richard Copin ◽

Mitchell J. Sullivan ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Ex Vivo ◽

Purine Biosynthesis ◽

In Vivo Studies ◽

Human Neutrophils ◽

Infection Models ◽

Genes Encoding ◽

Virulence Regulator

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR’s canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR. Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

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Staphylococcus epidermidis MSCRAMM SesJ Is Encoded in Composite Islands

mBio ◽

10.1128/mbio.02911-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Srishtee Arora ◽

Xiqi Li ◽

Andrew Hillhouse ◽

Kranti Konganti ◽

Sara V. Little ◽

...

Keyword(s):

Cell Wall ◽

Antibiotic Resistance ◽

Virulence Factors ◽

Staphylococcus Epidermidis ◽

Core Genome ◽

Clinical Isolates ◽

Modification System ◽

Content Type ◽

Genes Encoding ◽

Staphylococcal Cassette Chromosome

ABSTRACT Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates. IMPORTANCE S. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections.

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Prevalence of genes encoding extracellular virulence factors among meticillin-resistant Staphylococcus aureus isolates from the University Hospital, Olomouc, Czech Republic

Journal of Medical Microbiology ◽

10.1099/jmm.0.47413-0 ◽

2008 ◽

Vol 57 (4) ◽

pp. 403-410 ◽

Cited By ~ 22

Author(s):

P. Sauer ◽

J. Síla ◽

T. Štosová ◽

R. Večeřová ◽

P. Hejnar ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Czech Republic ◽

Virulence Factors ◽

Virulence Factor ◽

Antibiotic Susceptibility ◽

University Hospital ◽

Virulence Determinants ◽

Virulence Markers ◽

Genes Encoding ◽

The University

A rather fast and complicated progression of an infection caused by some strains of Staphylococcus aureus could be associated with the expression and co-action of virulence factor complexes in these strains. This study screened the antibiotic susceptibility and prevalence of virulence markers in isolates of meticillin-resistant S. aureus (MRSA) obtained from patients hospitalized at the University Hospital in Olomouc, Czech Republic. A total of 100 isolates was screened for 13 genes encoding extracellular virulence determinants (tst, pvl, eta, etb, sea, seb, sec, sed, see, seg, seh, sei and sej) and for their distribution in sample types. Eighty-nine isolates were positive for at least one of the genes. Genes for etb, pvl, see and seh were not detected in any of the MRSA isolates. No statistically significant differences in the occurrence of the determinants studied among sample types were found.

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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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