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.

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.

Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51.

Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPSC putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPSC in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPSC led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (epsT) in EPSC containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of epsT resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPSC that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios.

Metabolism of branched-chain amino acids revealed by transcriptome analysis in Vibrio alginolyticus.

Branched-chain amino acids (BCAAs) play important roles in nitrogen metabolism. However, little is known about the metabolism of BCAAs in the fish pathogen Vibrio alginolyticus. In this study, the global gene expression patterns of V. alginolyticus ZJ-T cultured in M63 minimal medium supplemented with ammonium sulfate or with three BCAAs (isoleucine, leucine and valine) as nitrogen source were evaluated by transcriptome analysis. The results revealed that 311 genes are up-regulated (|log2(Fold Change)|>1), which are involved in the pathways of flagellar assembly, bacterial chemotaxis and oxidative phosphorylation etc, and meanwhile 251 genes are down-regulated, which are involved in the pathways of BCAAs biosynthesis, selenocompound metabolism and C5-branced dibasic acid metabolism etc. This study contributes to the understanding of the BCAAs metabolism in the Vibrios.

Complete Genome Sequence of Vibrio alginolyticus ZJ-T.

Vibrio alginolyticus is a ubiquitous Gram-negative bacterium which is normally distributed in the coastal and estuarine environments. It has been suggested to be an opportunistic pathogen to both marine animals and humans, Here, the completed genome sequence of V. alginolyticus ZJ-T was determined by Illumina high-throughput sequencing.