Phenotypic characterization and virulence of a sae−agr− mutant of Staphylococcus aureus

A sae::Tn551 agr::tetM double mutant was constructed and characterized. The production of several exoproteins (e.g., β-hemolysin, DNase, and proteases) by this mutant was determined and found to be lower than the already diminished production of either isogenic single mutant sae− or agr−. The double mutant also showed, like the agr− mutant, null production of α- and δ-hemolysins and diminished levels of lipase. The reduced levels of many exoproteins in the double mutant as compared with their already diminished levels in either single mutant suggest that there is an additive or synergistic interaction between the two mutations involved, sae− and agr−. However, inactivation of both loci, sae and agr, had a different effect on the two exoproteins that are up regulated in the agr− mutant; thus, coagulase dropped to levels close to the null levels of the sae− parental strain, while extracellular protein A displayed the high levels characteristic of the agr− single mutant. The virulence of the sae−agr− double mutant, determined by intraperitoneal injection in mice, was found to be significantly diminished as compared with that of the sae+agr+ parental strain or the sae−agr+ single mutant.Key words: Staphylococcus aureus, exoprotein expression, sae− mutant, agr− mutant.

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Adherence of Staphylococcus aureus to Endothelial Cells: Influence of Capsular Polysaccharide, Global Regulatoragr, and Bacterial Growth Phase

Infection and Immunity ◽

10.1128/iai.68.9.4865-4871.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 4865-4871 ◽

Cited By ~ 89

Author(s):

Petra Pöhlmann-Dietze ◽

Martina Ulrich ◽

Kevin B. Kiser ◽

Gerd Döring ◽

Jean C. Lee ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Endothelial Cells ◽

Bacterial Growth ◽

Growth Phase ◽

Parental Strain ◽

Double Mutant ◽

Capsular Polysaccharide ◽

Stationary Growth Phase ◽

Exponential Growth Phase ◽

Logarithmic Phase

ABSTRACT The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase onS. aureus adherence to EC. Whereas S. aureusNewman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in theagr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC.S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P < 0.005). Complementation of the cap5Omutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant andcap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

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Effect of Ciprofloxacin on the Growth and Biofilm Formation Ability of Staphylococcus aureus

International Journal of Pathogen Research ◽

10.9734/ijpr/2021/v7i230181 ◽

2021 ◽

pp. 44-59

Author(s):

N. Queenette, Obinaju ◽

C. Chukwunonyerem, Ogwunga ◽

O. Sylvia, Anyadoh-Nwadike ◽

U. Emmanuel, Nwakwasi

Keyword(s):

Staphylococcus Aureus ◽

Defense Mechanism ◽

Bacterial Flora ◽

Protein A ◽

Inhibitory Effect ◽

Phenotypic Characterization ◽

Upper Respiratory Tract ◽

Staphylococcal Infections ◽

Superficial Skin ◽

Time Kill

Staphylococcus aureus is part of the normal bacterial flora of the skin, intestine and upper respiratory tract of both humans and animals and has the potential of causing staphylococcal infections if there is a breach in the hosts’ defense mechanism. These infections could range from mild superficial skin infections to more severe and even fatally invasive diseases such as sepsis and toxic shock syndrome. The infectivity of S. aureus is attributed to its ability to withstand extreme conditions and its possession of various virulence factors. The aim of this project was to study the effect of ciprofloxacin on the growth and biofilm forming ability of CM10 strain of Staphylococcus aureus using time kill study, resazurin and live/dead staining of biofilms and Real-time polymerase chain reaction. The identity of the given CM10 strain was confirmed when the result of the API-Staph was in total accordance with the results obtained from the colony morphology and phenotypic characterization tests (Coagulase/protein A, Gram, and Catalase tests). CM10 strain of S. aureus was not susceptible to 0.25mg/L of ciprofloxacin used for the time kill experiment but was susceptible to a minimum inhibitory concentration of 0.5mg/L. The difference between the ciprofloxacin treated biofilms of CM10 strain and the untreated biofilms was significant (P<0.05) showing that ciprofloxacin has an adverse effect on the cells in the biofilm. The results of this study provide an insight on the growth as well as the biofilm forming ability of CM10 strain of Staphylococcus aureus. Ciprofloxacin has been shown to be an effective antibacterial against this strain of S. aureus by its inhibitory effect on the growth as well as biofilm forming ability of this strain of S. aureus. This information would assist in developing novel anti-biofilm therapies to help in the management of biofilm mediated infections thereby reducing the morbidity and mortality rate of staphylococcal infections.

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SarS, a SarA Homolog Repressible by agr, Is an Activator of Protein A Synthesis in Staphylococcus aureus

Infection and Immunity ◽

10.1128/iai.69.4.2448-2455.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2448-2455 ◽

Cited By ~ 63

Author(s):

Ambrose L. Cheung ◽

Katherine Schmidt ◽

Brian Bateman ◽

Adhar C. Manna

Keyword(s):

Parental Strain ◽

Double Mutant ◽

Specific Binding ◽

Protein A ◽

Acid Protein ◽

Regulatory Loci ◽

Parental Level ◽

High Level ◽

Divergent Finding ◽

Amino Acid Protein

ABSTRACT The expression of protein A (spa) is repressed by global regulatory loci sarA and agr. Although SarA may directly bind to the spa promoter to downregulatespa expression, the mechanism by which agrrepresses spa expression is not clearly understood. In searching for SarA homologs in the partially released genome, we found a SarA homolog, encoding a 250-amino-acid protein designated SarS, upstream of the spa gene. The expression ofsarS was almost undetectable in parental strain RN6390 but was highly expressed in agr and sarA mutants, strains normally expressing high level of protein A. Interestingly, protein A expression was decreased in a sarS mutant as detected in an immunoblot but returned to near-parental levels in a complemented sarS mutant. Transcriptional fusion studies with a 158- and a 491-bp spa promoter fragment linked to the xylE reporter gene disclosed that the transcription of the spa promoter was also downregulated in thesarS mutant compared with the parental strain. Interestingly, the enhancement in spa expression in anagr mutant returned to a near-parental level in theagr sarS double mutant but not in the sarA sarSdouble mutant. Correlating with this divergent finding is the observation that enhanced sarS expression in anagr mutant was repressed by the sarA locus supplied in trans but not in a sarA mutant expressing RNAIII from a plasmid. Gel shift studies also revealed the specific binding of SarS to the 158-bp spa promoter. Taken together, these data indicated that the agr locus probably mediates spa repression by suppressing the transcription ofsarS, an activator of spa expression. However, the pathway by which the sarA locus downregulatesspa expression is sarS independent.

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Regulation of Exoprotein Gene Expression by the Staphylococcus aureus cvfB Gene

Infection and Immunity ◽

10.1128/iai.01552-06 ◽

2007 ◽

Vol 75 (4) ◽

pp. 1964-1972 ◽

Cited By ~ 26

Author(s):

Yasuhiko Matsumoto ◽

Chikara Kaito ◽

Daisuke Morishita ◽

Kenji Kurokawa ◽

Kazuhisa Sekimizu

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Virulence Factors ◽

Genetic Background ◽

Promoter Activity ◽

Double Mutant ◽

Gene Deletion ◽

Protein A ◽

Protease Production ◽

Gene Deletion Mutant

ABSTRACT We previously reported that the cvfB gene (SA1223) of Staphylococcus aureus is responsible for the virulence of this pathogenic bacterium. We show here that the cvfB gene regulates exoprotein gene expression. In a cvfB gene deletion mutant, hemolysin, DNase, and protease production were decreased, whereas protein A expression was increased. The amount of RNAIII, the transcript from the P3 promoter in the agr locus that regulates the expression of various virulence factors, was also reduced in the cvfB mutant. In addition, P2 and P3 promoter activity in the agr locus was decreased in the mutant. Under the genetic background of the agr-null mutation, cvfB gene disruption decreased the production levels of DNase and protease. Moreover, the cvfB and agr double mutant was less virulent than the agr mutant in silkworms. These results suggest that the cvfB gene product contributes to the expression of virulence factors and to pathogenicity via both agr-dependent and agr-independent pathways.

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