The Type 7b Secretion System of S. aureus and Its Role in Colonization and Systemic Infection.

Staphylococcus aureus bears a type 7b secretion system (T7bSS) that assembles in the bacterial envelope to promote the secretion of WXG-like proteins and toxic effectors bearing LXG domains. Cognate immunity proteins bind cytosolic effectors to mute their toxicity prior to secretion. T7b-secreted factors have been associated with the pathogenesis of staphylococcal disease and intraspecies competition. We identified earlier strain WU1, an S. aureus ST88 isolate that caused outbreaks of skin and soft tissue infections in mouse breeding facilities. WU1 was also found to persistently colonize the nasopharynx of animals, suggesting a strong host adaptation. In this manner, WU1 colonization and infectivity in mice resembles that of methicillin-sensitive and -resistant S. aureus strains in humans, where nasal carriage is a major risk factor for invasive infections. Here, animals were colonized with wild-type or T7-deficient WU1 strains or combinations thereof. Absence of the T7bSS did not affect colonization in the nasopharynx of animals, and although fluctuations were observed in weekly samplings, the wild-type strain did not replace the T7-deficient strain in cocolonization experiments. Bloodstream infection with a T7b-deficient strain resulted in enhanced survival and reduced bacterial loads and abscesses in soft tissues compared to infection with wild-type WU1. Together, experiments using a mouse-adapted strain suggest that the T7bSS of S. aureus is an important contributor to the pathogenesis of invasive disease.

Contribution of the EssC ATPase to the assembly of the type 7b secretion system in Staphylococcus aureus.

Secretion systems utilize ATPase activity to facilitate the translocation of proteins into and across membranes. In bacteria, the universally conserved SecA ATPase binds a large repertoire of preproteins and interacts with the SecYEG translocon. In contrast, the type 7b secretion system (T7bSS) of Staphylococcus aureus supports the secretion of a restricted subset of proteins. T7bSSs are found in several Firmicutes as gene clusters encoding secreted WXG100 proteins and FtsK/SpoIIIE-like ATPase. In S. aureus, this ATPase is called EssC and comprises two cytosolic forkhead-associated domains (FHA1-2), two membrane-spanning segments (TM1-2), and four cytosolic modules named DUF (domain of unknown function) and ATPases1-3 (D1D2D3). However, a detailed understanding of the interactions of EssC in the T7bSS is not clear. Here, we tagged EssC and performed affinity chromatography of detergent-solubilized extracts of wild type and isogenic mutants of S. aureus. We found that EssC recruits EsaA, EssA, and EssB in a complex referred to as the ESS (ESAT-6 like secretion system) translocon, and secreted substrates were not required for translocon assembly. Furthermore, deletions of FHA1 and DUF rendered EssC unstable, whereas FHA2 was required for association with EssB. This interaction was independent of EsaA, but EsaA was required to recruit EssA to the EssC-EssB complex. Finally, we show that assembly of the ESS translocon was impaired upon mutation of D2 structural motifs. Together, our data indicate that the ESS translocon is maintained fully assembled at the plasma membrane and that D2 is fundamental in sustaining the integrity of this complex.

Effects of individual base-pairs on in vivo target search and destruction kinetics of bacterial small RNA.

Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA-mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24-31% and increase koff by 14-25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.

Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS.

Small RNA (sRNA) regulators promote efficient responses to stress, but the mechanisms for prioritizing target mRNA regulation remain poorly understood. This study examines mechanisms underlying hierarchical regulation by the sRNA SgrS, found in enteric bacteria and produced under conditions of metabolic stress. SgrS posttranscriptionally coordinates a nine-gene regulon to restore growth and homeostasis. An in vivo reporter system quantified SgrS-dependent regulation of target genes and established that SgrS exhibits a clear target preference. Regulation of some targets is efficient even at low SgrS levels, whereas higher SgrS concentrations are required to regulate other targets. In vivo and in vitro analyses revealed that RNA structure and the number and position of base pairing sites relative to the start of translation impact the efficiency of regulation of SgrS targets. The RNA chaperone Hfq uses distinct modes of binding to different SgrS mRNA targets, which differentially influences positive and negative regulation. The RNA degradosome plays a larger role in regulation of some SgrS targets compared to others. Collectively, our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features and that the combination of these features precisely tunes the efficiency of regulation of multi-target sRNA regulons.

EssH Peptidoglycan Hydrolase Enables Staphylococcus aureus Type VII Secretion across the Bacterial Cell Wall Envelope.

The ESAT-6-like secretion system (ESS) of Staphylococcus aureus is assembled in the bacterial membrane from core components that promote the secretion of WXG-like proteins (EsxA, EsxB, EsxC, and EsxD) and the EssD effector. Genes encoding the ESS secretion machinery components, effector, and WXG-like proteins are located in the ess locus. Here, we identify essH, a heretofore uncharacterized gene of the ess locus, whose product is secreted via an N-terminal signal peptide into the extracellular medium of staphylococcal cultures. EssH exhibits two peptidoglycan hydrolase activities, cleaving the pentaglycine cross bridge and the amide bond of N-acetylmuramyl-l-alanine, thereby separating glycan chains and wall peptides with cleaved cross bridges. Unlike other peptidoglycan hydrolases, EssH does not promote the lysis of staphylococci. EssH residues Cys199 and His254, which are conserved in other CHAP domain enzymes, are required for peptidoglycan hydrolase activity and for S. aureus ESS secretion. These data suggest that EssH and its murein hydrolase activity are required for protein secretion by the ESS pathway.IMPORTANCE Gene clusters encoding WXG-like proteins and FtsK/SpoIIIE-like P loop ATPases in Firmicutes encode type 7b secretion systems (T7bSS) for the transport of select protein substrates. The Staphylococcus aureus T7bSS assembles in the bacterial membrane and promotes the secretion of WXG-like proteins and effectors. The mechanisms whereby staphylococci extend the T7SS across the bacterial cell wall envelope are not known. Here, we show that staphylococci secrete EssH to cleave their peptidoglycan, thereby enabling T7bSS transport of proteins across the bacterial cell wall envelope.

Diverse mechanisms of post-transcriptional repression by the small RNA regulator of glucose-phosphate stress.

The Escherichia coli small RNA SgrS controls a metabolic stress response that occurs upon accumulation of certain glycolytic intermediates. SgrS base pairs with and represses translation of ptsG and manXYZ mRNAs, which encode sugar transporters, and activates translation of yigL mRNA, encoding a sugar phosphatase. This study defines four new genes as direct targets of E. coli SgrS. These new targets, asd, adiY, folE and purR, encode transcription factors or enzymes of diverse metabolic pathways, including aspartate semialdehyde dehydrogenase, arginine decarboxylase gene activator, GTP cyclohydrolase I and a repressor of purine biosynthesis, respectively. SgrS represses translation of each of the four target mRNAs via distinct mechanisms. SgrS binding sites overlapping the Shine-Dalgarno sequences of adiY and folE mRNAs suggest that SgrS pairing with these targets directly occludes ribosome binding and prevents translation initiation. SgrS binding within the purR coding sequence recruits the RNA chaperone Hfq to directly repress purR translation. Two separate SgrS binding sites were found on asd mRNA, and both are required for full translational repression. Ectopic overexpression of asd, adiY and folE is specifically detrimental to cells experiencing glucose-phosphate stress, suggesting that SgrS-dependent repression of the metabolic functions encoded by these targets promotes recovery from glucose-phosphate stress.

Small RNAs Regulate Primary and Secondary Metabolism in Gram-negative Bacteria.

Over the last decade, small (often noncoding) RNA molecules have been discovered as important regulators influencing myriad aspects of bacterial physiology and virulence. In particular, small RNAs (sRNAs) have been implicated in control of both primary and secondary metabolic pathways in many bacterial species. This chapter describes characteristics of the major classes of sRNA regulators, and highlights what is known regarding their mechanisms of action. Specific examples of sRNAs that regulate metabolism in gram-negative bacteria are discussed, with a focus on those that regulate gene expression by base pairing with mRNA targets to control their translation and stability.

The small RNA SgrS: roles in metabolism and pathogenesis of enteric bacteria.

Bacteria adapt to ever-changing habitats through specific responses to internal and external stimuli that result in changes in gene regulation and metabolism. One internal metabolic cue affecting such changes in Escherichia coli and related enteric species is cytoplasmic accumulation of phosphorylated sugars such as glucose-6-phosphate or the non-metabolizable analog α-methylglucoside-6-phosphate. This "glucose-phosphate stress" triggers a dedicated stress response in γ-proteobacteria including several enteric pathogens. The major effector of this stress response is a small RNA (sRNA), SgrS. In E. coli and Salmonella, SgrS regulates numerous mRNA targets via base pairing interactions that result in alterations in mRNA translation and stability. Regulation of target mRNAs allows cells to reduce import of additional sugars and increase sugar efflux. SgrS is an unusual sRNA in that it also encodes a small protein, SgrT, which inhibits activity of the major glucose transporter. The two functions of SgrS, base pairing and production of SgrT, reduce accumulation of phosphorylated sugars and thereby relieve stress and promote growth. Examination of SgrS homologs in many enteric species suggests that SgrS has evolved to regulate distinct targets in different organisms. For example, in Salmonella, SgrS base pairs with sopD mRNA and represses production of the encoded effector protein, suggesting that SgrS may have a specific role in the pathogenesis of some γ-proteobacteria. In this review, we outline molecular mechanisms involved in SgrS regulation of its target mRNAs. We also discuss the response to glucose-phosphate stress in terms of its impact on metabolism, growth physiology, and pathogenesis.

Regulation of bacterial metabolism by small RNAs using diverse mechanisms.

Bacteria live in many dynamic environments with alternating cycles of feast or famine that have resulted in the evolution of mechanisms to quickly alter their metabolic capabilities. Such alterations often involve complex regulatory networks that modulate expression of genes involved in nutrient uptake and metabolism. A great number of protein regulators of metabolism have been characterized in depth. However, our ever-increasing understanding of the roles played by RNA regulators has revealed far greater complexity to regulation of metabolism in bacteria. Here, we review the mechanisms and functions of selected bacterial RNA regulators and discuss their importance in modulating nutrient uptake as well as primary and secondary metabolic pathways.

Computational analysis of bacterial RNA-Seq data.

Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of bacterial transcriptomes. However, computational methods for analysis of bacterial transcriptome data have not kept pace with the large and growing data sets generated by RNA-seq technology. Here, we present new algorithms, specific to bacterial gene structures and transcriptomes, for analysis of RNA-seq data. The algorithms are implemented in an open source software system called Rockhopper that supports various stages of bacterial RNA-seq data analysis, including aligning sequencing reads to a genome, constructing transcriptome maps, quantifying transcript abundance, testing for differential gene expression, determining operon structures and visualizing results. We demonstrate the performance of Rockhopper using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps generated by our algorithms are highly accurate when compared with focused experimental data from E. coli and N. gonorrhoeae, and we validate our system's ability to identify novel small RNAs, operons and transcription start sites. Our results suggest that Rockhopper can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes.