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1.
Cell Rep ; 43(4): 114082, 2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38583155

ABSTRACT

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteremia, and treatment failure is often associated with vancomycin-intermediate S. aureus isolates. The regulatory 3' UTR of the vigR mRNA contributes to vancomycin tolerance and upregulates the autolysin IsaA. Using MS2-affinity purification coupled with RNA sequencing, we find that the vigR 3' UTR also regulates dapE, a succinyl-diaminopimelate desuccinylase required for lysine and peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the 3' UTR increased virulence, while the isaA mutant is completely attenuated in a wax moth larvae model. Sequence and structural analyses of vigR indicated that the 3' UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions that contribute sequence for the isaA interaction seed and may functionalize the 3' UTR.


Subject(s)
3' Untranslated Regions , Staphylococcal Infections , Staphylococcus aureus , Animals , 3' Untranslated Regions/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Gene Expression Regulation, Bacterial , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Methicillin-Resistant Staphylococcus aureus/drug effects , Moths/microbiology , Staphylococcal Infections/microbiology , Staphylococcal Infections/drug therapy , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicity , Staphylococcus aureus/drug effects , Vancomycin/pharmacology , Virulence/genetics
2.
mSystems ; 9(4): e0097123, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38534138

ABSTRACT

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. IMPORTANCE: The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.


Subject(s)
Methicillin-Resistant Staphylococcus aureus , RNA, Small Untranslated , Staphylococcal Infections , Humans , Staphylococcus aureus/genetics , Vancomycin/pharmacology , Ribonuclease III , Methicillin-Resistant Staphylococcus aureus/genetics , RNA, Bacterial/genetics , RNA, Small Untranslated/genetics , Anti-Bacterial Agents/pharmacology , RNA, Messenger/genetics , Bacteria/genetics , Carbon
3.
Methods Enzymol ; 692: 299-324, 2023.
Article in English | MEDLINE | ID: mdl-37925184

ABSTRACT

Regulatory small RNA (sRNA) have been extensively studied in model Gram-negative bacteria, but the functional characterisation of these post-transcriptional gene regulators in Gram-positives remains a major challenge. Our previous work in enterohaemorrhagic E. coli utilised the proximity-dependant ligation technique termed CLASH (UV-crosslinking, ligation, and sequencing of hybrids) for direct high-throughput sequencing of the regulatory sRNA-RNA interactions within the cell. Recently, we adapted the CLASH technique and demonstrated that UV-crosslinking and RNA proximity-dependant ligation can be applied to Staphylococcus aureus, which uncovered the first RNA-RNA interaction network in a Gram-positive bacterium. In this chapter, we describe modifications to the CLASH technique that were developed to capture the RNA interactome associated with the double-stranded endoribonuclease RNase III in two clinical isolates of S. aureus. To briefly summarise our CLASH methodology, regulatory RNA-RNA interactions were first UV-crosslinked in vivo to the RNase III protein and protein-RNA complexes were affinity-purified using the His6-TEV-FLAG tags. Linkers were ligated to RNase III-bound RNA during library preparation and duplexed RNA-RNA species were ligated together to form a single contiguous RNA 'hybrid'. The RNase III-RNA binding sites and RNA-RNA interactions occurring on RNase III (RNA hybrids) were then identified by paired-end sequencing technology. RNase III-CLASH represents a step towards a systems-level understanding of regulatory RNA in Gram-positive bacteria.


Subject(s)
Escherichia coli Proteins , RNA, Small Untranslated , Endoribonucleases/genetics , Ribonuclease III/genetics , Ribonuclease III/metabolism , Staphylococcus aureus/genetics , Escherichia coli/genetics , Ribonuclease, Pancreatic , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Small Untranslated/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial
4.
Nat Commun ; 14(1): 1051, 2023 02 24.
Article in English | MEDLINE | ID: mdl-36828918

ABSTRACT

A new variant of Streptococcus pyogenes serotype M1 (designated 'M1UK') has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes 'M1global' and M1UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5' transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.


Subject(s)
Scarlet Fever , Streptococcal Infections , Humans , Streptococcus pyogenes/genetics , Scarlet Fever/epidemiology , Superantigens , Bacterial Proteins/genetics , United Kingdom , Exotoxins/genetics , Mutation , Australia
6.
Nat Commun ; 13(1): 3560, 2022 06 22.
Article in English | MEDLINE | ID: mdl-35732654

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.


Subject(s)
Methicillin-Resistant Staphylococcus aureus , RNA, Small Untranslated , Ribonuclease III , Gene Expression Regulation, Bacterial , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Untranslated/genetics , RNA, Small Untranslated/metabolism , Ribonuclease III/genetics , Ribonuclease III/metabolism
7.
Nat Commun ; 13(1): 3558, 2022 06 22.
Article in English | MEDLINE | ID: mdl-35732665

ABSTRACT

Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA-RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA-RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA-mRNA interactions are recovered and we find that an mRNA encoding a long 3' untranslated region (UTR) (termed vigR 3'UTR) functions as a regulatory 'hub' within the RNA-RNA interaction network. We demonstrate that the vigR 3'UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA-mRNA base-pairing. Deletion of the vigR 3'UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3'UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.


Subject(s)
Methicillin-Resistant Staphylococcus aureus , Ribonuclease III , Vancomycin Resistance , 3' Untranslated Regions/genetics , Anti-Bacterial Agents/therapeutic use , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/metabolism , Microbial Sensitivity Tests , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonuclease III/genetics , Ribonuclease III/metabolism , Vancomycin/pharmacology , Vancomycin Resistance/genetics
8.
mBio ; 12(4): e0104121, 2021 08 31.
Article in English | MEDLINE | ID: mdl-34372700

ABSTRACT

Regulatory RNAs have emerged as ubiquitous gene regulators in all bacterial species studied to date. The combination of sequence-specific RNA interactions and malleable RNA structure has allowed regulatory RNA to adopt different mechanisms of gene regulation in a diversity of genetic backgrounds. In the model Gammaproteobacteria Escherichia coli and Salmonella, the regulatory RNA chaperone Hfq appears to play a global role in gene regulation, directly controlling ∼20 to 25% of the entire transcriptome. While the model Firmicutes Bacillus subtilis and Staphylococcus aureus encode a Hfq homologue, its role has been significantly depreciated. These bacteria also have marked differences in RNA turnover. E. coli and Salmonella degrade RNA through internal endonucleolytic and 3'→5' exonucleolytic cleavage that appears to allow transient accumulation of mRNA 3' UTR cleavage fragments that contain stabilizing 3' structures. In contrast, B. subtilis and S. aureus are able to exonucleolytically attack internally cleaved RNA from both the 5' and 3' ends, efficiently degrading mRNA 3' UTR fragments. Here, we propose that the lack of 5'→3' exoribonuclease activity in Gammaproteobacteria has allowed the accumulation of mRNA 3' UTR ends as the "default" setting. This in turn may have provided a larger pool of unconstrained RNA sequences that has fueled the expansion of Hfq function and small RNA (sRNA) regulation in E. coli and Salmonella. Conversely, the exoribonuclease RNase J may be a significant barrier to the evolution of 3' UTR sRNAs in B. subtilis and S. aureus that has limited the pool of RNA ligands available to Hfq and other sRNA chaperones, depreciating their function in these model Firmicutes.


Subject(s)
Bacteria/genetics , Exoribonucleases/genetics , Exoribonucleases/metabolism , RNA Stability , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Bacteria/classification , Gammaproteobacteria/genetics , Gene Expression Regulation, Bacterial , Gram-Positive Bacteria/genetics , RNA, Bacterial/genetics
9.
Trends Genet ; 37(1): 35-45, 2021 01.
Article in English | MEDLINE | ID: mdl-32951948

ABSTRACT

The golden age of antibiotics has passed, and the threat of untreatable antimicrobial resistant infections is now a reality for many individuals. Understanding how bacteria resist antimicrobial treatment and regulate gene expression in response to antibiotics is an important step towards combating resistance. In this review we focus on a ubiquitous class of bacterial gene regulators termed regulatory small RNAs (sRNAs) and how they contribute to antimicrobial resistance and tolerance. Small RNAs have notable roles in modulating the composition of the bacterial envelope, and through these functions control intrinsic antimicrobial resistance in many human pathogens. Recent technical advances that allow profiling of the 'sRNA interactome' have revealed a complex post-transcriptional network of sRNA interactions that can be used to identify network hubs and regulatory bottlenecks. Sequence-specific inhibition of these sRNAs with programmable RNA-targeting therapeutics may present avenues for treating antimicrobial resistant pathogens or resensitizing to our current antibiotics.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/genetics , Drug Resistance, Microbial , Gene Expression Regulation, Bacterial , Gene Regulatory Networks , RNA, Bacterial/genetics , RNA, Small Untranslated/genetics , Animals , Bacteria/drug effects , Bacteria/growth & development , Humans
10.
BMC Genomics ; 19(1): 781, 2018 Oct 29.
Article in English | MEDLINE | ID: mdl-30373517

ABSTRACT

BACKGROUND: Bacterial filamentation occurs when rod-shaped bacteria grow without dividing. To identify genetically encoded inhibitors of division that promote filamentation, we used cell sorting flow cytometry to enrich filamentous clones from an inducible expression library, and then identified the cloned DNA with high-throughput DNA sequencing. We applied the method to an expression library made from fragmented genomic DNA of uropathogenic E. coli UTI89, which undergoes extensive reversible filamentation in urinary tract infections and might encode additional regulators of division. RESULTS: We identified 55 genomic regions that reproducibly caused filamentation when expressed from the plasmid vector, and then further localized the cause of filamentation in several of these to specific genes or sub-fragments. Many of the identified genomic fragments encode genes that are known to participate in cell division or its regulation, and others may play previously-unknown roles. Some of the prophage genes identified were previously implicated in cell division arrest. A number of the other fragments encoded potential short transcripts or peptides. CONCLUSIONS: The results provided evidence of potential new links between cell division and distinct cellular processes including central carbon metabolism and gene regulation. Candidate regulators of the UTI-associated filamentation response or others were identified amongst the results. In addition, some genomic fragments that caused filamentation may not have evolved to control cell division, but may have applications as artificial inhibitors. Our approach offers the opportunity to carry out in depth surveys of diverse DNA libraries to identify new genes or sequences encoding the capacity to inhibit division and cause filamentation.


Subject(s)
Bacteria/genetics , Cell Division/genetics , Gene Expression Regulation, Bacterial , Escherichia coli Proteins/genetics , Gene Library , High-Throughput Nucleotide Sequencing , Open Reading Frames , Phenotype , Uropathogenic Escherichia coli/genetics
11.
Article in English | MEDLINE | ID: mdl-28642845

ABSTRACT

Escherichia coli ordinarily resides in the lower gastrointestinal tract in humans, but some strains, known as Uropathogenic E. coli (UPEC), are also adapted to the relatively harsh environment of the urinary tract. Infections of the urine, bladder and kidneys by UPEC may lead to potentially fatal bloodstream infections. To survive this range of conditions, UPEC strains must have broad and flexible metabolic capabilities and efficiently utilize scarce essential nutrients. Whole-organism (or "omics") methods have recently provided significant advances in our understanding of the importance of metabolic adaptation in the success of UPECs. Here we describe the nutritional and metabolic requirements for UPEC infection in these environments, and focus on particular metabolic responses and adaptations of UPEC that appear to be essential for survival in the urinary tract.


Subject(s)
Adaptation, Physiological , Escherichia coli Infections/urine , Urinary Tract Infections/microbiology , Urinary Tract/microbiology , Uropathogenic Escherichia coli/metabolism , Uropathogenic Escherichia coli/pathogenicity , Amino Acids/metabolism , Anti-Infective Agents/pharmacology , Biomarkers , Carbon/metabolism , Escherichia coli Infections/diagnosis , Escherichia coli Proteins/drug effects , Escherichia coli Proteins/metabolism , Humans , Intestines/microbiology , Iron/metabolism , Kidney/microbiology , Metabolism , Metabolomics , Purines/metabolism , Pyrimidines/metabolism , Urinary Bladder/microbiology , Uropathogenic Escherichia coli/genetics , Uropathogenic Escherichia coli/growth & development , Virulence , Virulence Factors/metabolism
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