Abstract Yersinia pestis , the etiological agent of plague, persists in an enzootic cycle involving mammals and fleas, requiring constant outer membrane (OM) adaptation to disparate host environments. One such pathway involves the glycine zipper 2TM domain-containing protein SlyB, a central component of the OM stress response and PhoPQ virulence pathway. While the OM is critical for virulence, the role of the OM lipoprotein SlyB in Y. pestis ecology and pathogenesis remains unknown. We show by phylogenetic analyses that slyB paralogs expanded in environmental bacteria, whereas the canonical slyB gene was under negative selective pressure during Y. pestis speciation from Yersinia pseudotuberculosis . Using rodent and flea infection models recapitulating Y. pestis natural history, we demonstrate that SlyB is specifically required to resist the mammalian immune system at 37°C, including neutrophil-mediated antimicrobial activity during lymph node colonization, but is dispensable in septicemic plague in rodents. Strikingly, SlyB is not required for flea colonization and resistance to the antimicrobial-peptide-based immunity of arthropods at lower temperatures. SlyB-dependent OM stress tolerance reveals a mechanism by which Y. pestis establishes bubonic plague, in line with its critical lipopolysaccharide structural switch. Our findings identify SlyB as an evolutionarily fine-tuned component of the Y. pestis envelope that mediates immune escape upon infection of mammalian hosts through maintenance of structural integrity.

Staphylococcus aureus biofilms pose significant challenges in clinical settings due to their growing resistance to conventional antibiotics. While cold atmospheric plasma (CAP) is known for its bactericidal effects, its impact on biofilm architecture and physiology, and subsequent antimicrobial susceptibility remains poorly understood. This study investigates the effects of CAP treatment on S. aureus biofilms. We demonstrate that CAP not only reduces biofilm but also disrupts biofilm architecture and alters bacterial cell morphology. Transcriptomic analysis reveals distinct gene expression profiles following treatments with CAP and hydrogen peroxide, one of its active components, highlighting unique stress responses. We further show that CAP treatment of S. aureus grown on agar plates sensitizes bacteria to additional CAP exposure and induces genomic alterations involved in cell envelope integrity, defence mechanisms and DNA replication, recombination, and repair. Importantly, CAP treatment enhances bacterial susceptibility to the topical antibiotic for wound care mupirocin. These findings provide novel insights into the mechanisms underlying CAP-mediated biofilm control and offer a promising strategy to potentiate existing antimicrobial therapies.

We report the complete genome sequences of two valuable strains to investigate plague pathogenesis: (i) Yersinia pestis strain 6/69, which was isolated from a bubonic plague patient in Madagascar and contains pCD1, pMT1, and pPCP1 virulence plasmids, and (ii) the 6/69 strain cured of pPCP1.

Bacteria exhibit remarkable adaptability in response to selective pressures encountered during infection and antibiotic treatment. We characterize four Yersinia enterocolitica clonal isolates from successive bacteremia episodes that evolved within an elderly patient over 14 years. Their common evolution is characterized by a genome size reduction resulting in the loss of about a hundred genes and a so far undescribed deletion in the DNA gyrase gene gyrA conferring quinolone resistance. Third-generation cephalosporin resistance of the last isolate correlates with a truncation of OmpF in synergy with an increased production of BlaA and AmpC β-lactamases. A strong proteome remodeling of the isolates reveals a perturbed stringent response, as well as impaired metabolism which substantiate their severe growth defects in vitro, accounting for antibiotics tolerance and possibly therapeutic failure. This study documents previously unreported genetic and phenotypic changes associated with in-host adaptation of a pathogenic Yersinia species under antibiotic pressure.

Yersinia pestis has spilled over from wild rodent reservoirs to commensal rodents and humans, causing three historically recorded pandemics. Depletion in the copy number of the plasmid-encoded virulence gene pla occurred in later-dated strains of the first and second pandemics, yet the biological relevance of the pla deletion has been difficult to test. We identified modern Y. pestis strains that independently acquired the same pla depletion as ancient strains and herein show that excision of pla from the multicopy pPCP1 plasmid is accompanied by the integration of a separate full pPCP1 harboring pla into the single-copy pCD1 plasmid, reducing pla dosage. Moreover, we demonstrate that this depletion decreases the mortality of mice in models of bubonic plague but not in the pneumonic and septicemic forms of the disease. We hypothesize that pla depletion may have been selectively advantageous in bubonic plague, owing to rodent fragmentation after pandemic-induced mortality.

Global burden of infectious diseases and antimicrobial resistance are major public health issues calling for innovative control measures. Bacterial NAD kinase (NADK) is a crucial enzyme for production of NADP(H) and growth. In Staphylococcus aureus, NADK promotes pathogenesis by supporting production of key virulence determinants. Here, we find that knockdown of NADK by CRISPR interference sensitizes S. aureus to osmotic stress and to stresses induced by antibiotics targeting the envelop as well as replication, transcription and translation. Thus, NADK represents a promising target for the development of inhibitors which could be used in combination with current antibiotics.

Cold atmospheric plasma (CAP) is a promising complement to tissue repair and regenerative medicine approaches. CAP has therapeutic potential in infected cutaneous wounds by mechanisms which remain enigmatic. Here, CAP is shown to activate phagocyte NADPH oxidase complex NOX2. CAP induced increased intracellular reactive oxygen species, alleviated by NOX2 inhibitors. Genetic and pharmacological inhibitions of NOX2 in macrophages and bioengineered skin infected with Staphylococcus aureus and treated with CAP reduced intracellular oxidants and increased bacterial survival. CAP triggered Rac activation and phosphorylation of p40phox and p47phox required for NOX2 assembly and activity. Furthermore, CAP induced collagen I expression by fibroblasts. Infection and healing kinetics showed that murine skin wounds infected with S. aureus and treated with CAP are characterized by decreased bacterial burden, increased length of neoepidermis and extracellular matrix formation. Collectively, our findings identify mechanisms triggered by CAP that subdue infection and result in enhanced repair following skin injury.

Bloodstream infection is a major public health concern associated with high mortality and high healthcare costs worldwide. Bacteremia can trigger fatal sepsis whose prevention, diagnosis, and management have been recognized as a global health priority by the World Health Organization. Additionally, infection control is increasingly threatened by antimicrobial resistance, which is the focus of global action plans in the framework of a One Health response. In-depth knowledge of the infection process is needed to develop efficient preventive and therapeutic measures. The pathogenesis of bloodstream infection is a dynamic process resulting from the invasion of the vascular system by bacteria, which finely regulate their metabolic pathways and virulence factors to overcome the blood immune defenses and proliferate. In this review, we highlight our current understanding of determinants of bacterial survival and proliferation in the bloodstream and discuss their interactions with the molecular and cellular components of blood.

Emerging and reemerging infectious diseases represent major public health concerns. The urgent need for infection control measures requires deep understanding of molecular pathogenesis. Global approaches to study biological systems such as mass-spectrometry based proteomics benefited from groundbreaking physical and bioinformatical technological developments over recent years. However, dual proteomic study of highly pathogenic microorganisms and their hosts in complex matrices encountered during infection remains challenging due to high protein dynamic range of samples and requirements imposed in biosafety level 3 or 4 laboratories. Here, we constructed a dual proteomic pipeline of Yersinia pestis in human blood and plasma, mirroring bacteremic phase of plague. We provide the most complete Y. pestis proteome revealing a major reshaping of important bacterial path-ways such as methionine biosynthesis and iron acquisition in human plasma. Remarkably, proteomic profiling in human blood highlights a greater Yersinia outer proteins intoxication of monocytes than neutrophils. Our study unravels global expression changes and points to a specific pathogenic signature during infection, paving the way for future exploration of proteomes in the complex context of host-pathogen interactions. Subject Categories Microbiology, Virology and Host Pathogen Interaction, Proteomics

We report the complete genome sequence of Yersinia pseudotuberculosis strain SP-1303, identified as part of lineage 8 and associated with Far East scarlet-like fever. The genome includes the chromosome, the Yersinia-virulence plasmid (pYV) encoding a type III secretion system essential for virulence, the pVM82 plasmid, and two cryptic plasmids.

The expanding genus Yersinia is composed of multiple nonpathogenic species and a few pathogenic species, including the deadly etiologic agent of plague, Yersinia pestis . In 2 decades, the number of genomic, transcriptomic, and proteomic studies on Yersinia grew massively, delivering a wealth of data.

AbstractEmerging and reemerging infectious diseases represent major public health concerns. The urgent need for infection control measures requires deep understanding of molecular pathogenesis. Global approaches to study biological systems such as mass-spectrometry based proteomics benefited from groundbreaking physical and bioinformatical technological developments over recent years. However, dual proteomic study of highly pathogenic microorganisms and their hosts in complex matrices encountered during infection remains challenging due to high protein dynamic range of samples and requirements imposed in biosafety level 3 or 4 laboratories. Here, we constructed a dual proteomic pipeline ofYersinia pestisin human blood and plasma, mirroring bacteremic phase of plague. We provide the most completeY. pestisproteome revealing a major reshaping of important bacterial path-ways such as methionine biosynthesis and iron acquisition in human plasma. Remarkably, proteomic profiling in human blood highlights a greaterYersiniaouter proteins intoxication of monocytes than neutrophils. Our study unravels global expression changes and points to a specific pathogenic signature during infection, paving the way for future exploration of proteomes in the complex context of host-pathogen interactions.Subject CategoriesMicrobiology, Virology and Host Pathogen Interaction, Proteomics

Here, we report complete genome sequences of two clinical isolates of Staphylococcus aureus, namely, Xen31 and Xen36, which have been genetically modified to express an optimized Photorhabdus luminescens luciferase operon. Xen31 and Xen36 are bioluminescent strains used widely for investigation of bacterial pathogenesis, drug discovery, and development of novel therapies.

Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus . Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection.

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection of E. coli genomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, is preferentially distributed in four common sequence types (ST) encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion of cnf1-positive multidrug-resistant ST131 strains from subclade H30Rx/C2, accounting for a rising prevalence of cnf1-positive strains in ST131. Despite the absence of phylogeographical signals, cnf1-positive isolates segregated into clusters in the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. The suggested dominant expansion of cnf1-positive strains in ST131-H30Rx/C2 led us to uncover the competitive advantage conferred by cnf1 for gut colonization to the clinical strain EC131GY ST131-H30Rx/C2 versus cnf1-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function of cnf1 was confirmed for another clinical strain ST131-H30Rx/C2. In addition, functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2 and a cnf1-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-H30Rx/C2 and suggested expansion of cnf1-positive MDR isolates in subclade ST131-H30Rx/C2.

Nicotinamide adenine dinucleotide kinases (NAD kinases) are essential and ubiquitous enzymes involved in the production of NADP(H) which is an essential cofactor in many metabolic pathways. Targeting NAD kinase (NADK), a rate limiting enzyme of NADP biosynthesis pathway, represents a new promising approach to treat bacterial infections. Previously, we have produced the first NADK inhibitor active against staphylococcal infection. From this linear di-adenosine derivative, namely NKI1, we designed macrocyclic analogues. Here, we describe the synthesis and evaluation of an original series of cyclic diadenosine derivatives as NADK inhibitors of two pathogenic bacteria, Listeria monocytogenes and Staphylococcus aureus. The nature and length of the link between the two adenosine units were examined leading to sub-micromolar inhibitors of NADK1 from L. monocytogenes, including its most potent in vitro inhibitor reported so far (with a 300-fold improvement compared to NKI1).