Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Airway environment drives the selection of quorum sensing mutants and promote Staphylococcus aureus chronic lifestyle.

Staphylococcus aureus is a predominant cause of chronic lung infections. While the airway environment is rich in highly sialylated mucins, the interaction of S. aureus with sialic acid is poorly characterized. Using S. aureus USA300 as well as clinical isolates, we demonstrate that quorum-sensing dysfunction, a hallmark of S. aureus adaptation, correlates with a greater ability to consume free sialic acid, providing a growth advantage in an air-liquid interface model and in vivo. Furthermore, RNA-seq experiment reveals that free sialic acid triggers transcriptional reprogramming promoting S. aureus chronic lifestyle. To support the clinical relevance of our results, we show the co-occurrence of S. aureus, sialidase-producing microbiota and free sialic acid in the airway of patients with cystic fibrosis. Our findings suggest a dual role for sialic acid in S. aureus airway infection, triggering virulence reprogramming and driving S. aureus adaptive strategies through the selection of quorum-sensing dysfunctional strains.

Genomic analysis of Staphylococcus aureus sequential isolates from lungs of patients with cystic fibrosis.

Staphylococcus aureus is the predominant pathogen in children with cystic fibrosis (CF) in France and, around 80% of them harbored S. aureus in their lungs. This study investigated virulence and antimicrobial resistance-associated genes and within-host evolution polymorphisms in 14 S. aureus persistent clones from 14 chronically infected CF children. For each of the 14 patients, we compared genomes of two isogenic sequential isolates separated by 2-9 years. All isolates were methicillin-sensitive and harbored the immune evasion gene cluster, whereas half of them harbored the enterotoxin gene cluster. Most clones were capsule type 8 (8/14) and accessory gene regulator (agr)-specificity group 1 (9/14). We identified convergent mutations in genes involved in carbohydrate metabolism, cell wall metabolism, genetic information processing and adhesion, which are likely to play important role in intracellular invasion and persistence. Further explorations relying notably on proteomics will contribute to improve our understanding of the mechanisms at play in the striking long-term persistence ability of S. aureus.

Lung-Adapted Staphylococcus aureus Isolates With Dysfunctional Agr System Trigger a Proinflammatory Response.

BACKGROUND: Staphylococcus aureus dominates the lung microbiota of children with cystic fibrosis (CF) and persistent clones are able to establish chronic infection for years, having a direct deleterious impact on lung function. However, in this context, the exact contribution of S. aureus to the decline in respiratory function in children with CF is not elucidated. METHODS: To investigate the contribution of persistent S. aureus clones in CF disease, we undertook the analysis of sequential isogenic isolates recovered from 15 young CF patients. RESULTS: Using an air-liquid infection model, we observed a strong correlation between S. aureus adaption in the lung (late isolates), low toxicity, and proinflammatory cytokine secretion. Conversely, early isolates appeared to be highly cytotoxic but did not promote cytokine secretion. We found that cytokine secretion was dependent on staphylococcal protein A (Spa), which was selectively expressed in late compared to early isolates as a consequence of dysfunctional agr quorum-sensing system. Finally, we demonstrated the involvement of TNF-α receptor 1 signaling in the inflammatory response of airway epithelial cells to these lung-adapted S. aureus isolates. CONCLUSIONS: Our results suggest an unexpected direct role of bacterial lung adaptation in the progression of chronic lung disease by promoting a proinflammatory response through acquired agr dysfunction.

A Role for Taok2 in Listeria monocytogenes Vacuolar Escape.

The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.

BLI-MS: Combining biolayer interferometry and mass spectrometry.

Biolayer interferometry (BLI) is a technology which allows to study the affinity between two interacting macro-molecules and to visualize their kinetic of interaction in real time. In this work, we combine BLI interaction measurement with mass spectrometry in order to identify the proteins interacting with the bait. We provide for the first time the proof of concept of the feasibility of BLI-MS in complex biological mixtures.

The pentose phosphate pathway constitutes a major metabolic hub in pathogenic Francisella.

Metabolic pathways are now considered as intrinsic virulence attributes of pathogenic bacteria and thus represent potential targets for antibacterial strategies. Here we focused on the role of the pentose phosphate pathway (PPP) and its connections with other metabolic pathways in the pathophysiology of Francisella novicida. The involvement of the PPP in the intracellular life cycle of Francisella was first demonstrated by studying PPP inactivating mutants. Indeed, we observed that inactivation of the tktA, rpiA or rpe genes severely impaired intramacrophage multiplication during the first 24 hours. However, time-lapse video microscopy demonstrated that rpiA and rpe mutants were able to resume late intracellular multiplication. To better understand the links between PPP and other metabolic networks in the bacterium, we also performed an extensive proteo-metabolomic analysis of these mutants. We show that the PPP constitutes a major bacterial metabolic hub with multiple connections to glycolysis, the tricarboxylic acid cycle and other pathways, such as fatty acid degradation and sulfur metabolism. Altogether our study highlights how PPP plays a key role in the pathogenesis and growth of Francisella in its intracellular niche.

Reactive Oxygen Species-Dependent Innate Immune Mechanisms Control Methicillin-Resistant Staphylococcus aureus Virulence in the Drosophila Larval Model.

Antibiotic-resistant Staphylococcus aureus strains constitute a major public health concern worldwide and are responsible for both health care- and community-associated infections. Here, we establish a robust and easy-to-implement model of oral S. aureus infection using Drosophila melanogaster larvae that allowed us to follow the fate of S. aureus at the whole-organism level as well as the host immune responses. Our study demonstrates that S. aureus infection triggers H2O2 production by the host via the Duox enzyme, thereby promoting antimicrobial peptide production through activation of the Toll pathway. Staphylococcal catalase mediates H2O2 neutralization, which not only promotes S. aureus survival but also minimizes the host antimicrobial response, hence reducing bacterial clearance in vivo. We show that while catalase expression is regulated in vitro by the accessory gene regulatory system (Agr) and the general stress response regulator sigma B (SigB), it no longer depends on these two master regulators in vivo. Finally, we confirm the versatility of this model by demonstrating the colonization and host stimulation capabilities of S. aureus strains belonging to different sequence types (CC8 and CC5) as well as of two other bacterial pathogens, Salmonella enterica serovar Typhimurium and Shigella flexneri. Thus, the Drosophila larva can be a general model to follow in vivo the innate host immune responses triggered during infection by human pathogens. IMPORTANCE The pathogenicity of methicillin-resistant S. aureus (MRSA) strains relies on their ability to produce a wide variety of tightly regulated virulence factors. Current in vivo models to analyze host-pathogen interactions are limited and difficult to manipulate. Here, we have established a robust and reliable model of oral S. aureus infection using Drosophila melanogaster larvae. We show that S. aureus stimulates host immunity through the production of reactive oxygen species (ROS) and antimicrobial peptide (AMP) and that ROS potentialize AMP gene expression. S. aureus catalase plays a key role in this complex environment and acts in vivo independently from SigB and Agr control. We propose that fly larvae can provide a general model for studying the colonization capabilities of human pathogens.

High-Resolution Typing of Staphylococcus epidermidis Based on Core Genome Multilocus Sequence Typing To Investigate the Hospital Spread of Multidrug-Resistant Clones.

Staphylococcus epidermidis is a pathogen emerging worldwide as a leading cause of health care-associated infections. A standardized high-resolution typing method to document transmission and dissemination of multidrug-resistant S. epidermidis strains is needed. Our aim was to provide a core genome multilocus sequence typing (cgMLST) scheme for S. epidermidis to improve the international surveillance of S. epidermidis We defined a cgMLST scheme based on 699 core genes and used it to investigate the population structure of the species and the genetic relatedness of isolates recovered from infants hospitalized in several wards of a French hospital. Our results show the long-lasting endemic persistence of S. epidermidis clones within and across wards of hospitals and demonstrate the ability of our cgMLST approach to identify and track these clones. We made the scheme publicly available through the Institut Pasteur BIGSdb server (http://bigsdb.pasteur.fr/epidermidis/). This tool should enable international harmonization of the epidemiological surveillance of multidrug-resistant S. epidermidis clones. By comparing gene distribution among infection and commensal isolates, we also confirmed the association of the mecA locus with infection isolates and of the fdh gene with commensal isolates. (This study has been registered at ClinicalTrials.gov under registration no. NCT03374371.).

Which Current and Novel Diagnostic Avenues for Bacterial Respiratory Diseases?

Bacterial acute pneumonia is responsible for an extremely large burden of death worldwide and diagnosis is paramount in the management of patients. While multidrug-resistant bacteria is one of the biggest health threats in the coming decades, clinicians urgently need access to novel diagnostic technologies. In this review, we will first present the already existing and largely used techniques that allow identifying pathogen-associated pneumonia. Then, we will discuss the latest and most promising technological advances that are based on connected technologies (artificial intelligence-based and Omics-based) or rapid tests, to improve the management of lung infections caused by pathogenic bacteria. We also aim to highlight the mutual benefits of fundamental and clinical studies for a better understanding of lung infections and their more efficient diagnostic management.

Multitasking Actors of Staphylococcus aureus Metabolism and Virulence.

Recent studies have uncovered the striking commonality of multitasking molecular actors linking metabolism and virulence regulation. Beyond the well known importance of carbohydrate and amino acid metabolism regulators in coordinating metabolic pathways and pathogenesis, we highlight recent major advances linking lipid and nucleic acid pathways to virulence regulation in Staphylococcus aureus.

Pivotal Role of Mitochondria in Macrophage Response to Bacterial Pathogens.

Mitochondria are essential organelles that act as metabolic hubs and signaling platforms within the cell. Numerous mitochondrial functions, including energy metabolism, lipid synthesis, and autophagy regulation, are intimately linked to mitochondrial dynamics, which is shaped by ongoing fusion and fission events. Recently, several intracellular bacterial pathogens have been shown to modulate mitochondrial functions to maintain their replicative niche. Through selected examples of human bacterial pathogens, we will discuss how infection induces mitochondrial changes in infected macrophages, triggering modifications of the host metabolism that lead to important immunological reprogramming.

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System.

The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis indeed identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. Substitutions of Y139 with alanine or phosphomimetics prevented T6SS formation and abolished phagosomal escape whereas substitution with phenylalanine delayed but did not abolish phagosomal escape in J774-1 macrophages. Altogether our data demonstrated that the Y139 site of IglB plays a critical role in T6SS biogenesis, suggesting that sheath phosphorylation could participate to T6SS dynamics.Data are available via ProteomeXchange with identifier PXD013619; and on MS-Viewer, key lkaqkllxwx.

Transketolase of Staphylococcus aureus in the Control of Master Regulators of Stress Response During Infection.

Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. The importance of the pentose phosphate pathway (PPP) during S. aureus infection is currently largely unexplored. In the current study, we focused on one key PPP enzyme, transketolase (TKT). We showed that inactivation of the unique gene encoding TKT activity in S. aureus USA300 (∆tkt) led to drastic metabolomic changes. Using time-lapse video imaging and mice infection, we observed a major defect of the ∆tkt strain compared with wild-type strain in early intracellular proliferation and in the ability to colonize kidneys. Transcriptional activity of the 2 master regulators sigma B and RpiRc was drastically reduced in the ∆tkt mutant during host cells invasion. The concomitant increased RNAIII transcription suggests that TKT-or a functional PPP-strongly influences the ability of S. aureus to proliferate within host cells by modulating key transcriptional regulators.

Multiscale NMR investigations of two anatomically contrasted genotypes of sorghum under watered conditions and during drought stress.

Today, in the presence of global warming, understanding how plants respond to drought stress is essential to meet the challenge of developing new cultivars and new irrigation strategies, consistent with the maintenance of crop productivity. In this context, the study of the relation between plants and water is of central interest for modeling their responses to biotic and abiotic constraints. Paradoxically, there are very few direct and noninvasive methods to quantify and measure the level and the flow of water in plants. The present work aims to develop a noninvasive methodology for living plant based on nuclear magnetic resonance (NMR) at low magnetic field and imaging (MRI) to tackle the issue of water quantity in plants. For this purpose, a portable NMR device measuring the signal level at 8 mT was built. This instrument addresses specific challenges such as miniaturization, accessibility, and overheating in order to maintain the plant intact of time over long period. Time dependence of the water content in sorghum plants is reported under abiotic stress as well as the fraction of transpirable soil water and the photosynthesis activity through the leaves. At high magnetic field (9.4 T), T2 maps were acquired on the same sorghum plants at two time points. The combination of these approaches allows us to identify ecophysiological biomarkers of drought stress. One particular interesting result concerns the spatial distribution of water in two anatomically contrasted sorghum genotypes.

Fulminant arterial vasculitis as an unusual complication of disseminated staphylococcal disease due to the emerging CC1 methicillin-susceptible Staphylococcus aureus clone: a case report.

BACKGROUND: Staphylococcus aureus has emerged as a leading cause of invasive severe diseases with a high rate of morbidity and mortality worldwide. The wide spectrum of clinical manifestations and outcome observed in staphylococcal illness may be a consequence of both microbial factors and variability of the host immune response. CASE PRESENTATION: A 14-years old child developed limb ischemia with gangrene following S. aureus bloodstream infection. Histopathology revealed medium-sized arterial vasculitis. The causing strain belonged to the emerging clone CC1-MSSA and numerous pathogenesis-related genes were identified. Patient's genotyping revealed functional variants associated with severe infections. A combination of virulence and host factors might explain this unique severe form of staphylococcal disease. CONCLUSION: A combination of virulence and genetic host factors might explain this unique severe form of staphylococcal disease.

Chronic Staphylococcus aureus Lung Infection Correlates With Proteogenomic and Metabolic Adaptations Leading to an Increased Intracellular Persistence.

BACKGROUND: Chronic lung infection in cystic fibrosis (CF) patients by Staphylococcus aureus is a well-established epidemiological fact. Indeed, S. aureus is the most commonly identified pathogen in the lungs of CF patients. Improving our understanding of the mechanisms associated with the persistence of S. aureus is therefore an important issue. METHODS: We selected pairs of sequential S. aureus isolates from 3 patients with CF and from 1 patient with non-CF chronic lung disease. We used a combination of genomic, proteomic, and metabolomic approaches with functional assays for in-depth characterization of S. aureus long-term persistence. RESULTS: In this study, we show that late S. aureus isolates from CF patients have an increased ability for intracellular survival in CF bronchial epithelial-F508del cells compared to ancestral early isolates. Importantly, the increased ability to persist intracellularly was confirmed for S. aureus isolates within the own-patient F508del epithelial cells. An increased ability to form biofilm was also demonstrated. Furthermore, we identified the underlying genetic modifications that induce altered protein expression profiles and notable metabolic changes. These modifications affect several metabolic pathways and virulence regulators that could constitute therapeutic targets. CONCLUSIONS: Our results strongly suggest that the intracellular environment might constitute an important niche of persistence and relapse necessitating adapted antibiotic treatments.

A splenic IgM memory subset with antibacterial specificities is sustained from persistent mucosal responses.

To what extent immune responses against the gut flora are compartmentalized within mucosal tissues in homeostatic conditions remains a much-debated issue. We describe here, based on an inducible AID fate-mapping mouse model, that systemic memory B cell subsets, including mainly IgM+ B cells in spleen, together with IgA+ plasma cells in spleen and bone marrow, are generated in mice in the absence of deliberate immunization. While the IgA component appears dependent on the gut flora, IgM memory B cells are still generated in germ-free mice, albeit to a reduced extent. Clonal relationships and renewal kinetics after anti-CD20 treatment reveal that this long-lasting splenic population is mainly sustained by output of B cell clones persisting in mucosal germinal centers. IgM-secreting hybridomas established from splenic IgM memory B cells showed reactivity against various bacterial isolates and endogenous retroviruses. Ongoing activation of B cells in gut-associated lymphoid tissues thus generates a diversified systemic compartment showing long-lasting clonal persistence and protective capacity against systemic bacterial infections.

The absence of N-acetylglucosamine in wall teichoic acids of Listeria monocytogenes modifies biofilm architecture and tolerance to rinsing and cleaning procedures.

The wall teichoic acid (WTA) is the major carbohydrate found within the extracellular matrix of the Listeria monocytogenes biofilm. We first addressed the frequency of spontaneous mutations in two genes (lmo2549 and lmo2550) responsible for the GlcNAcylation in 93 serotype 1/2a strains that were mainly isolated from seafood industries. We studied the impact of mutations in lmo2549 or lmo2550 genes on biofilm formation by using one mutant carrying a natural mutation inactivating the lmo2550 gene (DSS 1130 BFA2 strain) and two EGD-e mutants that lack respective genes by in-frame deletion of lmo2549 or lmo2550 using splicing-by-overlap-extension PCR, followed by allelic exchange mutagenesis. The lmo2550 gene mutation, occurring in around 50% isolates, caused a decrease in bacterial adhesion to stainless steel compared to wild-type EGD-e strain during the adhesion step. On the other hand, bacterial population weren't significantly different after 24h-biofilm formation. The biofilm architecture was different between the wild-type strain and the two mutants inactivated for lmo2549 or lmo2550 genes respectively with the presence of bacterial micro-colonies for mutants which were not observed in the wild-type EGD-e strain biofilm. These differences might account for the stronger hydrophilic surface exhibited by the mutant cells. Upon a water flow or to a cleaning procedure at a shear stress of 0.16 Pa, the mutant biofilms showed the higher detachment rate compared to wild-type strain. Meanwhile, an increase in the amount of residual viable but non-culturable population on stainless steel was recorded in two mutants. Our data suggests that the GlcNAc residue of WTA played a role in adhesion and biofilm formation of Listeria monocyctogenes.

Antibacterial Toxins: Gram-Positive Bacteria Strike Back!

Bacteria live in communities where strains compete with each other by deploying an arsenal of antibacterial toxins. While the past decade revealed the vast array of antibacterial toxins secreted by Gram-negative bacteria, several recent studies have begun to uncover the ability of Gram-positive bacteria to battle with their own weapons.