The accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these ...opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci.
We generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to form a biofilm in the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard.
The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host.
The DNA-dependent RNA polymerase core enzyme in Gram-positive bacteria consists of seven subunits. Whilst four of them (α2ββ′) are essential, three smaller subunits, δ, ɛ and ω (∼9–21.5 kDa), are ...considered accessory. Both δ and ω have been viewed as integral components of RNAP for several decades; however, ɛ has only recently been described. Functionally these three small subunits carry out a variety of tasks, imparting important, supportive effects on the transcriptional process of Gram-positive bacteria. While ω is thought to have a wide range of roles, reaching from maintaining structural integrity of RNAP to σ factor recruitment, the only suggested function for ɛ thus far is in protecting cells from phage infection. The third subunit, δ, has been shown to have distinct influences in maintaining transcriptional specificity, and thus has a key role in cellular fitness. Collectively, all three accessory subunits, although dispensable under laboratory conditions, are often thought to be crucial for proper RNAP function. Herein we provide an overview of the available literature on each subunit, summarizing landmark findings that have deepened our understanding of these proteins and their function, and outline future challenges in understanding the role of these small subunits in the transcriptional process.
This review details over four decades of research on the small, accessory subunits of the Gram-positive transcriptional machinery.
It is becoming increasingly apparent that Staphylococcus aureus are able to survive engulfment by macrophages, and that the intracellular environment of these host cells, which is essential to innate ...host defenses against invading microorganisms, may in fact provide a refuge for staphylococcal survival and dissemination. Based on this, we postulated that S. aureus might induce cytoprotective mechanisms by changing gene expression profiles inside macrophages similar to obligate intracellular pathogens, such as Mycobacterium tuberculosis. To validate our hypothesis we first ascertained whether S. aureus infection could affect programmed cell death in human (hMDMs) and mouse (RAW 264.7) macrophages and, specifically, protect these cells against apoptosis. Our findings indicate that S. aureus-infected macrophages are more resistant to staurosporine-induced cell death than control cells, an effect partly mediated via the inhibition of cytochrome c release from mitochondria. Furthermore, transcriptome analysis of human monocyte-derived macrophages during S. aureus infection revealed a significant increase in the expression of antiapoptotic genes. This was confirmed by quantitative RT-PCR analysis of selected genes involved in mitochondria-dependent cell death, clearly showing overexpression of BCL2 and MCL1. Cumulatively, the results of our experiments argue that S. aureus is able to induce a cytoprotective effect in macrophages derived from different mammal species, which can prevent host cell elimination, and thus allow intracellular bacterial survival. Ultimately, it is our contention that this process may contribute to the systemic dissemination of S. aureus infection.
(
), an emerging opportunistic pathogen, predominantly infects individuals with underlying pulmonary diseases such as cystic fibrosis (CF). Current treatment outcomes for
infections are poor due to
...inherent antibiotic resistance and unique host interactions that promote phenotypic tolerance and hinder drug access. The hypoxic, mucus-laden airways in the CF lung and antimicrobial phagosome within macrophages represent hostile niches
must overcome
alterations in gene expression for survival. Regulatory mechanisms important for the adaptation and long-term persistence of
within the host are poorly understood, warranting further genetic and transcriptomics study of this emerging pathogen. DosRS
, a two-component signaling system (TCS), is one proposed mechanism utilized to subvert host defenses and counteract environmental stress such as hypoxia. The homologous TCS of
(
), DosRS
, is known to induce a ~50 gene regulon in response to hypoxia, carbon monoxide (CO) and nitric oxide (NO)
and
. Previously, a small DosR
regulon was predicted using bioinformatics based on DosR
motifs however, the role and regulon of DosRS
in
pathogenesis have yet to be characterized in depth. To address this knowledge gap, our lab generated a
knockout strain (
to investigate differential gene expression, and phenotype in an
hypoxia model of dormancy. qRT-PCR and lux reporter assays demonstrate
and 6 predicted downstream genes are induced in hypoxia. In addition, RNAseq revealed induction of a much larger hypoxia response comprised of >1000 genes, including 127 differentially expressed genes in a
mutant strain. Deletion of DosRS
led to attenuated growth under low oxygen conditions, a shift in morphotype from smooth to rough, and down-regulation of 216 genes. This study provides the first look at the global transcriptomic response of
to low oxygen conditions encountered in the airways of CF patients and within macrophage phagosomes. Our data also demonstrate the importance of DosRS
for adaptation of
to hypoxia, highlighting a distinct regulon (compared to
that is significantly larger than previously described, including both genes conserved across mycobacteria as well as
-specific genes.
is a major cause of skin and soft tissue infection. The bacterium expresses four major proteases that are emerging as virulence factors: aureolysin (Aur), V8 protease (SspA), staphopain A (ScpA), and ...staphopain B (SspB). We hypothesized that human galectin-3, a β-galactoside-binding lectin involved in immune regulation and antimicrobial defense, is a target for these proteases and that proteolysis of galectin-3 is a novel immune evasion mechanism. Indeed, supernatants from laboratory strains and clinical isolates of
caused galectin-3 degradation. Similar proteolytic capacities were found in
isolates but not in
Galectin-3-induced activation of the neutrophil NADPH oxidase was abrogated by bacterium-derived proteolysis of galectin-3, and SspB was identified as the major protease responsible. The impact of galectin-3 and protease expression on
virulence was studied in a murine skin infection model. In galectin-3
mice, SspB-expressing
caused larger lesions and resulted in higher bacterial loads than protease-lacking bacteria. No such difference in bacterial load or lesion size was detected in galectin-3
mice, which overall showed smaller lesion sizes than the galectin-3
animals. In conclusion, the staphylococcal protease SspB inactivates galectin-3, abrogating its stimulation of oxygen radical production in human neutrophils and increasing tissue damage during skin infection.
Mutation of sarA in Staphylococcus aureus results in a reduced capacity to form a biofilm, but the mechanistic basis for this remains unknown. Previous transcriptional profiling experiments ...identified a number of genes that are differentially expressed both in a biofilm and in a sarA mutant. This included genes involved in acid tolerance and the production of nucleolytic and proteolytic exoenzymes. Based on this we generated mutations in alsSD, nuc and sspA in the S. aureus clinical isolate UAMS-1 and its isogenic sarA mutant and assessed the impact on biofilm formation. Because expression of alsSD was increased in a biofilm but decreased in a sarA mutant, we also generated a plasmid construct that allowed expression of alsSD in a sarA mutant. Mutation of alsSD limited biofilm formation, but not to the degree observed with the corresponding sarA mutant, and restoration of alsSD expression did not restore the ability to form a biofilm. In contrast, concomitant mutation of sarA and nuc significantly enhanced biofilm formation by comparison to the sarA mutant. Although mutation of sspA had no significant impact on the ability of a sarA mutant to form a biofilm, a combination of protease inhibitors (E-64, 1-10-phenanthroline, and dichloroisocoumarin) that was shown to inhibit the production of multiple extracellular proteases without inhibiting growth was also shown to enhance the ability of a sarA mutant to form a biofilm. This effect was evident only when all three inhibitors were used concurrently. This suggests that the reduced capacity of a sarA mutant to form a biofilm involves extracellular proteases of all three classes (serine, cysteine and metalloproteases). Inclusion of protease inhibitors also enhanced biofilm formation in a sarA/nuc mutant, with the combined effect of mutating nuc and adding protease inhibitors resulting in a level of biofilm formation with the sarA mutant that approached that of the UAMS-1 parent strain. These results demonstrate that the inability of a sarA mutant to repress production of extracellular nuclease and multiple proteases have independent but cumulative effects that make a significant contribution to the biofilm-deficient phenotype of an S. aureus sarA mutant.
In this study, we identify a novel two-component system in
(herein named AmsSR for regulator of alternative metabolic systems) only present in select gammaproteobacterial and betaproteobacterial ...species. Bioinformatic analysis revealed that the histidine kinase, AmsS, contains 14 predicted N-terminal transmembrane domains and harbors a hybrid histidine kinase arrangement in its C-terminus. Transcriptional analysis revealed the proton ionophore CCCP selectively induces P
expression. Disruption of
resulted in decreased intracellular pH and increased depolarization of cytoplasmic membranes. Transcriptome profiling revealed a major reordering of metabolic circuits upon
disruption, with energy generation pathways typically used by bacteria growing in limited oxygen being favored. Interestingly, we observed enhanced growth rates for mutant strains in the presence of glucose, which led to overproduction of pyruvate. To mitigate the toxic effects of carbon overflow, we noted acetate overproduction in
-null strains, resulting from a hyperactive Pta-AckA pathway. Additionally, due to altered expression of key metabolic genes,
mutants favor an incomplete TCA cycle, relying heavily on an overactive glyoxylate shunt. This metabolic reordering overproduces NADH, which is not oxidized by the ETC; components of which were significantly downregulated upon
disruption. As a result, the mutants almost exclusively rely on substrate phosphorylation for ATP production, and consequently display reduced oxygen consumption in the presence of glucose. Collectively, our data suggests that disruption of
affects the function of the aerobic respiratory chain, impacting the energy status of the cell, which in turn upregulates alternative metabolic and energy generation pathways.
controls the progression of infection through the coordinated production of extracellular proteases, which selectively modulate virulence determinant stability. This is evidenced by our previous ...finding that a protease-null strain has a hypervirulent phenotype in a murine model of sepsis, resulting from the unchecked accumulation of virulence factors. Here, we dissect the individual roles of these proteases by constructing and assessing the pathogenic potential of a combinatorial protease mutant library. When strains were constructed bearing increasing numbers of secreted proteases, we observed a variable impact on infectious capacity, where some exhibited hypervirulence, while others phenocopied the wild-type. The common thread for hypervirulent strains was that each lacked both aureolysin and staphopain A. Upon assessment, we found that the combined loss of these two enzymes alone was necessary and sufficient to engender hypervirulence. Using proteomics, we identified a number of important secreted factors, including SPIN, LukA, Sbi, SEK, and PSMα4, as well as an uncharacterized chitinase-related protein (SAUSA300_0964), to be overrepresented in both the
and the protease-null mutants. When assessing the virulence of
SAUSA300_0964 and
mutants, we found that hypervirulence was completely eliminated, whereas
and
strains elicited aggressive infections akin to the protease double mutant. Collectively, our findings shed light on the influence of extracellular proteases in controlling the infectious process and identifies SAUSA300_0964 as an important new component of the
virulence factor arsenal.
A key feature of the pathogenic success of
is the myriad virulence factors encoded within its genome. These are subject to multifactorial control, ensuring their timely production only within an intended infectious niche. A key node in this network of control is the secreted proteases of
, who specifically and selectively modulate virulence factor stability. In our previous work we demonstrated that deletion of all 10 secreted proteases results in hypervirulence, since virulence factors exist unchecked, leading to overly aggressive infections. Here, using a combinatorial collection of protease mutants, we reveal that deletion of aureolysin and staphopain A is necessary and sufficient to elicit hypervirulence. Using proteomic techniques, we identify the collection of virulence factors that accumulate in hypervirulent protease mutants, and demonstrate that a well-known toxin (LukA) and an entirely novel secreted element (SAUSA300_0964) are the leading contributors to deadly infections observed in protease-lacking strains.
The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as “nutritional immunity.” Staphylococcus aureus growth is ...inhibited by calprotectin in vitro, and calprotectin is localized in vivo to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of S. aureus and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of S. aureus during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in clpP, which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of S. aureus to the presence of calprotectin. ClpP contributes to pathogenesis in vivo in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for S. aureus to outcompete the host for metals.IMPORTANCEStaphylococcus aureus is a leading cause of skin and soft tissue infections, bloodstream infections, and endocarditis. Antibiotic treatment failures during S. aureus infections are increasingly prevalent, highlighting the need for novel antimicrobial agents. Metal chelator-based therapeutics have tremendous potential as antimicrobials due to the strict requirement for nutrient metals exhibited by bacterial pathogens. The high-affinity transition metal-binding properties of calprotectin represents a potential therapeutic strategy that functions through metal chelation. Our studies provide a foundation to define mechanisms by which S. aureus combats nutritional immunity and may be useful for the development of novel therapeutics to counter the ability of S. aureus to survive in a metal-limited environment.
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