Anti-TNF Agents Restrict Adherent-invasive Escherichia coli Replication Within Macrophages Through Modulation of Chitinase 3-like 1 in Patients with Crohn's Disease.

BACKGROUND AND AIMS: The mechanism of action of anti-tumour necrosis factor [anti-TNF] agents could implicate macrophage modulation in Crohn's disease [CD]. As CD macrophages are defective in controlling CD-associated adherent-invasive Escherichia coli [AIEC], anti-TNF agents could limit AIEC replication within macrophages. We assessed the effect of anti-TNF agents on AIEC survival within monocyte-derived macrophages [MDMs] from CD patients and attempted to identify the proteins involved. METHODS: Peripheral blood MDMs were obtained from 44 CD patients [22 with and 22 without anti-TNF agents]. MDMs were infected with reference strain AIEC-LF82. Proteomic analysis was performed before and 6 h after AIEC-LF82 infection. RESULTS: AIEC-LF82 survival was lower in MDMs from CD patients receiving anti-TNF agents compared to those who did not [-73%, p = 0.006]. After AIEC-LF82 infection, the levels of CD82 [p = 0.007], ILF3 [Interleukin enhancer-binding factor 3; p = 0.001], FLOT-1 [Flotillin-1; p = 0.007] and CHI3L1 [Chitinase 3-like 1; p = 0.035] proteins were different within CD-MDMs depending on anti-TNF exposure. FLOT-1 [ϱ = -0.44; p = 0.038] and CHI3L1 [ϱ = 0.57, p = 0.006] levels were inversely and positively correlated with AIEC survival within MDMs from CD patients with or without anti-TNF, respectively. We observed a dose-dependent decrease of AIEC-LF82 survival after adjunction of anti-TNF within MDMs, inducing an increase of FLOT-1 and decrease of CHI3L1 mRNA levels. Neutralization of intra-macrophagic CHI3L1 protein using anti-CHI3L1 antibodies reduced AIEC survival within macrophages 6 h after infection [p < 0.05]. CONCLUSION: Anti-TNF agents are able to restrict replication of pathobionts, such as AIEC, within macrophages by modulating FLOT-1 and CHI3L1 expression in CD patients.

Deep impact of the inactivation of the SecA2-only protein export pathway on the proteosurfaceome of Listeria monocytogenes.

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and ΔsecA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and surface properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. SIGNIFICANCE: L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology.

Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa.

In recent years, the effectiveness of antimicrobials in the treatment of Pseudomonas aeruginosa infections has gradually decreased. This pathogen can be observed in several clinical cases, such as pneumonia, urinary tract infections, sepsis, in immunocompromised hosts, such as neutropenic cancer, burns, and AIDS patients. Furthermore, Pseudomonas aeruginosa causes diseases in both livestock and pets. The highly flexible and versatile genome of P. aeruginosa allows it to have a high rate of pathogenicity. The numerous secreted virulence factors, resulting from its numerous secretion systems, the multi-resistance to different classes of antibiotics, and the ability to produce biofilms are pathogenicity factors that cause numerous problems in the fight against P. aeruginosa infections and that must be better understood for an effective treatment. Infections by P. aeruginosa represent, therefore, a major health problem and, as resistance genes can be disseminated between the microbiotas associated with humans, animals, and the environment, this issue needs be addressed on the basis of an One Health approach. This review intends to bring together and describe in detail the molecular and metabolic pathways in P. aeruginosa's pathogenesis, to contribute for the development of a more targeted therapy against this pathogen.

Exoproteomic analysis of the SecA2-dependent secretion in Listeria monocytogenes EGD-e.

As part of the Sec translocase, the accessory ATPase SecA2 is present in some pathogenic Gram-positive bacteria. In Listeria monocytogenes, deletion of secA2 results in filamentous cells that form rough colonies and have lower virulence. However, only a few proteins have been identified that are secreted by this pathway. This investigation aims to provide the first exoproteomic analysis of the SecA2-dependent secretion in L. monocytogenes EGD-e. By using media and temperatures relevant to bacterial physiology, we demonstrated that the rough colony and elongated bacterial cell morphotypes are highly dependent on growth conditions. Subsequently, comparative exoproteomic analyses of the ΔsecA2 versus wt strains were performed in chemically defined medium at 20°C and 37°C. Analyzing the proteomic data following the secretomics-based method, part of the proteins appeared routed towards the Sec pathway and exhibited an N-terminal signal peptide. For another significant part, they were primarily cytoplasmic proteins, thus lacking signal peptide and with no predictable secretion pathway. In total, 13 proteins were newly identified as secreted via SecA2, which were essentially associated with cell-wall metabolism, adhesion and/or biofilm formation. From this comparative exoproteomic analysis, new insights into the L. monocytogenes physiology are discussed in relation to its saprophytic and pathogenic lifestyle.

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure.

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.