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Bacteria respond to stress by rapidly regulating gene expression. Regulation can occur through the control of messenger RNA (mRNA) production (transcription elongation), stability of mRNAs, or translation of mRNAs. Bacteria can use small RNAs (sRNAs) to regulate gene expression at each of these steps, but we often do not understand how this works at a molecular level. In this study, we find that sRNAs in Escherichia coli regulate gene expression at the level of transcription elongation by promoting or inhibiting transcription termination by a protein called Rho. These results help us understand new molecular mechanisms of gene expression regulation in bacteria.

Invasive Staphylococcus aureus infections cause high morbidity and mortality in children and adults. With rising antimicrobial resistance, optimal prevention strategies and novel therapeutics are needed. As an effective vaccine remains elusive, characterization of invasive isolates over time is required to identify determinants of invasive infection. S. aureus isolates recovered from children with invasive infection and those with colonization were obtained. Isolates were examined by whole genome sequencing to evaluate gene repertoire, sequence type, clonal complex, and phylogenetic characterization, and isolate characteristics were correlated to clinical data. 118 children with invasive S. aureus infections were enrolled; 56% of infections were caused by methicillin-susceptible S. aureus (MSSA). Methicillin-resistance (MRSA) was associated with increased inflammation, though clinical outcomes of MRSA vs MSSA did not differ. Colonization isolates exhibited higher sequence type diversity than invasive isolates. Nine distinct clonal complexes (CC) were identified among all isolates; CC8 and CC5 were associated with higher clinical severity scores. Accessory gene regulator locus type 1, Panton-Valentine Leukocidin, and arginine catabolic mobile element declined over time. Staphylokinase and leukocidin ED were associated with invasive infection, while enterotoxin B was more frequent in colonizing isolates. We observed a significant expansion in sequence type diversity among invasive clinical isolates over 12 years with the emergence of newly invasive clones in recent years. The presence of staphylokinase and LukED were associated with invasive infection over time. These findings provide insights into the pathogenesis of invasive S. aureus and may provide putative targets for immunologic approaches to prevention.

USA300 has remained the dominant community and healthcare associated methicillin-resistant (MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American epidemic (SAE) clade and North American epidemic (NAE) clade. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Here, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome, to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage. Phylogenetic analysis with these genomes confirms that the NAE and SAE USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We provide evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South and North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC , which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus , blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a Δ fumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation. This study reveals how the pathogen Staphylococcus aureus adapts to the lung microenvironment, rich in host metabolites fumarate and itaconate, by using a key enzyme, FumC, to support its metabolic fitness, survival, and persistence.

Background The Covid-19 pandemic has been characterized by the emergence of novel SARS-CoV-2 variants, each with distinct properties influencing transmission dynamics, immune escape, and virulence, which, in turn, influence their impact on local populations. Swift analysis of the properties of newly emerged variants is essential in the initial days and weeks to enhance readiness and facilitate the scaling of clinical and public health system responses. Methods This paper introduces a two-variant metapopulation compartmental model of disease transmission to simulate the dynamics of disease transmission during a period of transition to a newly dominant strain. Leveraging novel S-gene dropout analysis data and genomic sequencing data, combined with confirmed Covid-19 case data, we estimate the epidemiological characteristics of the Omicron variant, which replaced the Delta variant in late 2021 in Philadelphia, PA. We utilized a grid-search method to identify plausible combinations of model parameters, followed by an ensemble adjustment Kalman filter for parameter inference. Results The model successfully estimated key epidemiological parameters; we estimated the ascertainment rate of 0.22 (95% credible interval 0.15–0.29) and transmission rate of 5.0 (95% CI 2.4–6.6) for the Omicron variant. Conclusions The study demonstrates the potential for this model-inference framework to provide real-time insights during the emergence of novel variants, aiding in timely public health responses.

SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25–3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

BackgroundInvasive Staphylococcus aureus infections cause high morbidity and mortality in children and adults. With rising antimicrobial resistance, optimal prevention strategies and novel therapeutics are needed. As an effective vaccine remains elusive, characterization of invasive isolates over time is required to identify determinants of invasive infection.MethodsS. aureus isolates recovered from children with invasive infection and those with colonization were obtained. Isolates were examined by whole genome sequencing to evaluate gene repertoire, sequence type, clonal complex, and phylogenetic characterization, and isolate characteristics were correlated to clinical data.Results118 children with invasive S. aureus infections were enrolled; 56% of infections were caused by methicillin-susceptible S. aureus (MSSA). Methicillin-resistance (MRSA) was associated with increased inflammation, though clinical outcomes of MRSA vs MSSA did not differ. Colonization isolates exhibited higher sequence type diversity than invasive isolates. Nine distinct clonal complexes (CC) were identified among all isolates; CC8 and CC5 were associated with higher clinical severity scores. Accessory gene regulator locus type 1, Panton-Valentine Leukocidin, and arginine catabolic mobile element declined over time. Staphylokinase and leukocidin ED were associated with invasive infection, while enterotoxin B was more frequent in colonizing isolates.ConclusionsWe observed a significant expansion in sequence type diversity among invasive clinical isolates over 12 years with the emergence of newly invasive clones in recent years. The presence of staphylokinase and LukED were associated with invasive infection over time. These findings provide insights into the pathogenesis of invasive S. aureus and may provide putative targets for immunologic approaches to prevention.

Bacteria use a multi-layered regulatory strategy to precisely and rapidly tune gene expression in response to environmental cues. Small RNAs (sRNAs) form an important layer of gene expression control and most act post-transcriptionally to control translation and stability of mRNAs. We have shown that at least five different sRNAs in Escherichia coli regulate the cyclopropane fatty acid synthase (cfa) mRNA. These sRNAs bind at different sites in the long 5' untranslated region (UTR) of cfa mRNA and previous work suggested that they modulate RNase E-dependent mRNA turnover. Recently, the cfa 5' UTR was identified as a site of Rho-dependent transcription termination, leading us to hypothesize that the sRNAs might also regulate cfa transcription elongation. In this study we find that a pyrimidine-rich region flanked by sRNA binding sites in the cfa 5' UTR is required for premature Rho-dependent termination. We discovered that both the activating sRNA RydC and repressing sRNA CpxQ regulate cfa primarily by modulating Rho-dependent termination of cfa transcription, with only a minor effect on RNase E-mediated turnover of cfa mRNA. A stem-loop structure in the cfa 5' UTR sequesters the pyrimidine-rich region required for Rho-dependent termination. CpxQ binding to the 5' portion of the stem increases Rho-dependent termination whereas RydC binding downstream of the stem decreases termination. These results reveal the versatile mechanisms sRNAs use to regulate target gene expression at transcriptional and post-transcriptional levels and demonstrate that regulation by sRNAs in long UTRs can involve modulation of transcription elongation.Competing Interest StatementThe authors have declared no competing interest.Footnotes* New data clarifying the mechanism of regulation of cfa mRNA by RydC and CpxQ sRNAs were added (new Fig. 6). The author who completed this work has been added (Andrew K. Buechler).

The small RNA (sRNA) RydC strongly activates cfa, which encodes the cyclopropane fatty acid synthase. Previous work demonstrated that RydC activation of cfa increases conversion of unsaturated fatty acids to cyclopropanated fatty acids in membrane lipids and changes the biophysical properties of membranes, making cells more resistant to acid stress. The conditions and regulators that control RydC synthesis had not previously been identified. In this study, we demonstrate that RydC regulation of cfa is important for resistance to membrane-disrupting conditions. We identify a GntR-family transcription factor, YieP, that represses rydC transcription and show that YieP indirectly regulates cfa through RydC. YieP positively autoregulates its own transcription. We further identify additional sRNA regulatory inputs that contribute to control of RydC and cfa. The translation of yieP is repressed by the Fnr-dependent sRNA, FnrS, making FnrS an indirect activator of rydC and cfa. Conversely, RydC activity on cfa is antagonized by the OmpR-dependent sRNA OmrB. Altogether, this work illuminates a complex regulatory network involving transcriptional and post-transcriptional inputs that link control of membrane biophysical properties to multiple environmental signals.

Abstract We report the genome of a B.1.1.7+E484K severe acute respiratory syndrome coronavirus 2 from Southeastern Pennsylvania and compare it with all high-coverage B.1.1.7+E484K genomes (n = 235) available. Analyses showed the existence of at least 4 distinct clades of this variant circulating in the United States and the possibility of at least 59 independent acquisitions of the E484K mutation.