CARD9 in neutrophils protects from colitis and controls mitochondrial metabolism and cell survival.

OBJECTIVES: Inflammatory bowel disease (IBD) results from a combination of genetic predisposition, dysbiosis of the gut microbiota and environmental factors, leading to alterations in the gastrointestinal immune response and chronic inflammation. Caspase recruitment domain 9 (Card9), one of the IBD susceptibility genes, has been shown to protect against intestinal inflammation and fungal infection. However, the cell types and mechanisms involved in the CARD9 protective role against inflammation remain unknown. DESIGN: We used dextran sulfate sodium (DSS)-induced and adoptive transfer colitis models in total and conditional CARD9 knock-out mice to uncover which cell types play a role in the CARD9 protective phenotype. The impact of Card9 deletion on neutrophil function was assessed by an in vivo model of fungal infection and various functional assays, including endpoint dilution assay, apoptosis assay by flow cytometry, proteomics and real-time bioenergetic profile analysis (Seahorse). RESULTS: Lymphocytes are not intrinsically involved in the CARD9 protective role against colitis. CARD9 expression in neutrophils, but not in epithelial or CD11c+cells, protects against DSS-induced colitis. In the absence of CARD9, mitochondrial dysfunction increases mitochondrial reactive oxygen species production leading to the premature death of neutrophilsthrough apoptosis, especially in oxidative environment. The decreased functional neutrophils in tissues might explain the impaired containment of fungi and increased susceptibility to intestinal inflammation. CONCLUSION: These results provide new insight into the role of CARD9 in neutrophil mitochondrial function and its involvement in intestinal inflammation, paving the way for new therapeutic strategies targeting neutrophils.

Reducing neutrophil exposure to oxygen allows their basal state maintenance.

Neutrophils are the most abundant circulating white blood cells and are the central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1-4% O2 ), to which they are well adapted. Neutrophils are atypical cells since they are highly glycolytic and susceptible to oxygen exposure, which induces their activation and death through mechanisms that remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxia. Here, we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. We demonstrate that neutrophil hyper-activation (CD62L shedding) is induced during culture under hyperoxic conditions (24 h), compared with neutrophils cultured under anoxic conditions. Spontaneous neutrophil extracellular trap (NET) formation is observed when neutrophils face hyperoxia during purification or culture. In addition, we show that maintaining neutrophils in autologous plasma is the preferred strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 57 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophil activation (CD11bhigh , CD54high , CD62Lneg ) or viability (DAPI+ ). We identified two clusters of proteins belonging to cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen exposure during neutrophil purification. In conclusion, protecting neutrophil from oxygen during their purification and culture is recommended to avoid activation and to prevent the alteration of cytosolic protein composition.

The role of Acinetobacter baumannii response regulator BfmR in pellicle formation and competitiveness via contact-dependent inhibition system.

BACKGROUND: Acinetobacter baumannii is one of the most important opportunistic pathogens responsible for hospital acquired infections. It displays multi-drug resistance profile and has the ability to colonize surfaces and persist under harsh conditions. A. baumannii two-component signal transduction system BfmRS, consisting of response regulator BfmR and sensor kinase BfmS, has been implicated in the control of various virulence-related traits and has been suggested to act as a global modulator of A. baumannii physiology. RESULTS: Here, we assessed the role of BfmR regulator in pellicle formation and bacterial competition, features important for the establishment of A. baumannii in clinical environment. We show that BfmR is required for the pellicle formation of A. baumannii, as ΔbfmRS mutant lacked this phenotype. The loss of bfmRS also greatly reduced the secretion of A. baumannii Hcp protein, which is a component of T6SS secretion system. However, T6SS-mediated killing phenotype was not impaired in ΔbfmRS mutant. On the contrary, the same mutation resulted in the transcriptional activation of contact-dependent inhibition (CDI) system, which A. baumannii used to inhibit the growth of another clinical A. baumannii strain and a closely related species Acinetobacter baylyi. CONCLUSIONS: The obtained results indicate that BfmR is not only required for the pellicle phenotype induction in A. baumannii, but also, due to the down-regulation of a CDI system, could allow the incorporation of other A. baumannii strains or related species, possibly increasing the likelihood of the pathogens' survival.

Blp1 protein shows virulence-associated features and elicits protective immunity to Acinetobacter baumannii infection.

BACKGROUND: Multidrug resistant Acinetobacter baumannii is one of the major infection agents causing nosocomial pneumonia. Therefore, new therapeutic approaches against this bacterium are needed. Surface-exposed proteins from bacterial pathogens are implicated in a variety of virulence-related traits and are considered as promising candidates for vaccine development. RESULTS: We show in this study that a large Blp1 protein from opportunistic pathogen A. baumannii is encoded in all examined clinical strains of globally spread international clonal lineages I (IC I) and II (IC II). The two blp1 gene variants exhibit lineage-specific distribution profile. By characterization of blp1 deletion mutants and their complementation with blp1 alleles we show that blp1 gene is required for A. baumannii biofilm formation and adhesion to epithelial cells in IC I strain but not in the IC II strain. Nevertheless both alleles are functional in restoring the deficient phenotypes of IC I strain. Moreover, the blp1 gene is required for the establishing of A. baumannii virulence phenotype in nematode and murine infection models. Additionally, we demonstrate that C-terminal 711 amino acid fragment of Blp1 elicits an efficient protection to lethal A. baumannii infection in a murine model using active and passive immunization approaches. Antiserum obtained against Blp1-specific antigen provides opsonophagocytic killing of A. baumannii in vitro. CONCLUSIONS: Lineage-specific variants of surface-exposed components of bacterial pathogens complicate the development of new therapeutic approaches. Though we demonstrated different impact of Blp1 variants on adherence of IC I and IC II strains, Blp1-specific antiserum neutralized A. baumannii strains of both clonal lineages. Together with the observed increased survival rate in vaccinated mice these results indicate that A. baumannii Blp1 protein could be considered as a new vaccine candidate.

The Mutation of Conservative Asp268 Residue in the Peptidoglycan-Associated Domain of the OmpA Protein Affects Multiple Acinetobacter baumannii Virulence Characteristics.

Acinetobacter baumannii is a nosocomial human pathogen of increasing concern due to its multidrug resistance profile. The outer membrane protein A (OmpA) is an abundant bacterial cell surface component involved in A. baumannii pathogenesis. It has been shown that the C-terminal domain of OmpA is located in the periplasm and non-covalently associates with the peptidoglycan layer via two conserved amino acids, thereby anchoring OmpA to the cell wall. Here, we investigated the role of one of the respective residues, D268 in OmpA of A. baumannii clinical strain Ab169, on its virulence characteristics by complementing the ΔompA mutant with the plasmid-borne ompAD268A allele. We show that while restoring the impaired biofilm formation of the ΔompA strain, the Ab169ompAD268A mutant tended to form bacterial filaments, indicating the abnormalities in cell division. Moreover, the Ab169 OmpA D268-mediated association to peptidoglycan was required for the manifestation of twitching motility, desiccation resistance, serum-induced killing, adhesion to epithelial cells and virulence in a nematode infection model, although it was dispensable for the uptake of ß-lactam antibiotics by outer membrane vesicles. Overall, the results of this study demonstrate that the OmpA C-terminal domain-mediated association to peptidoglycan is critical for a number of virulent properties displayed by A. baumannii outside and within the host.

Distinct regulation of the anterior and posterior myeloperoxidase expression by Etv2 and Gata1 during primitive Granulopoiesis in zebrafish.

Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.

Neutrophils: from IBD to the gut microbiota.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract that results from dysfunction in innate and/or adaptive immune responses. Impaired innate immunity, which leads to lack of control of an altered intestinal microbiota and to activation of the adaptive immune system, promotes a secondary inflammatory response that is responsible for tissue damage. Neutrophils are key players in innate immunity in IBD, but their roles have been neglected compared with those of other immune cells. The latest studies on neutrophils in IBD have revealed unexpected complexities, with heterogeneous populations and dual functions, both deleterious and protective, for the host. In parallel, interconnections between disease development, intestinal microbiota and neutrophils have been highlighted. Numerous IBD susceptibility genes (such as NOD2, NCF4, LRRK2, CARD9) are involved in neutrophil functions related to defence against microorganisms. Moreover, severe monogenic diseases involving dysfunctional neutrophils, including chronic granulomatous disease, are characterized by intestinal inflammation that mimics IBD and by alterations in the intestinal microbiota. This observation demonstrates the dialogue between neutrophils, gut inflammation and the microbiota. Neutrophils affect microbiota composition and function in several ways. In return, microbial factors, including metabolites, regulate neutrophil production and function directly and indirectly. It is crucial to further investigate the diverse roles played by neutrophils in host-microbiota interactions, both at steady state and in inflammatory conditions, to develop new IBD therapies. In this Review, we discuss the roles of neutrophils in IBD, in light of emerging evidence proving strong interconnections between neutrophils and the gut microbiota, especially in an inflammatory context.

A polyamine acetyltransferase regulates the motility and biofilm formation of Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen highly resistant to environmental changes and antimicrobial treatments. Regulation of cellular motility and biofilm formation is important for its virulence, although it is poorly described at the molecular level. It has been previously reported that Acinetobacter genus specifically produces a small positively charged metabolite, polyamine 1,3-diaminopropane, that has been associated with cell motility and virulence. Here we show that A. baumannii encodes novel acetyltransferase, Dpa, that acetylates 1,3-diaminopropane, directly affecting the bacterium motility. Expression of dpa increases in bacteria that form pellicle and adhere to eukaryotic cells as compared to planktonic bacterial cells, suggesting that cell motility is linked to the pool of non-modified 1,3-diaminopropane. Indeed, deletion of dpa hinders biofilm formation and increases twitching motion confirming the impact of balancing the levels of 1,3-diaminopropane on cell motility. The crystal structure of Dpa reveals topological and functional differences from other bacterial polyamine acetyltransferases, adopting a ß-swapped quaternary arrangement similar to that of eukaryotic polyamine acetyltransferases with a central size exclusion channel that sieves through the cellular polyamine pool. The structure of catalytically impaired DpaY128F in complex with the reaction product shows that binding and orientation of the polyamine substrates are conserved between different polyamine-acetyltransferases.

Ascorbate deficiency increases progression of shigellosis in guinea pigs and mice infection models.

Shigella spp. are the causative agents of bacterial dysentery and shigellosis, mainly in children living in developing countries. The study of Shigella entire life cycle in vivo and the evaluation of vaccine candidates' protective efficacy have been hampered by the lack of a suitable animal model of infection. None of the studies evaluated so far (rabbit, guinea pig, mouse) allowed the recapitulation of full shigellosis symptoms upon Shigella oral challenge. Historical reports have suggested that dysentery and scurvy are both metabolic diseases associated with ascorbate deficiency. Mammals, which are susceptible to Shigella infection (humans, non-human primates and guinea pigs) are among the few species unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate deficiency, but not scurvy, in guinea pigs to investigate whether poor vitamin C status increases the progression of shigellosis. Moderate ascorbate deficiency increased shigellosis symptom severity during an extended period of time (up to 48 h) in all strains tested (Shigella sonnei, Shigella flexneri 5a, and 2a). At late time points, an important influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although Shigella was able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. Moreover, we found that ascorbate deficiency also increased Shigella penetration into the colon epithelium layer in a Gulo-/- mouse infection model. The use of these new rodent models of shigellosis opens new doors for the study of both Shigella infection strategies and immune responses to Shigella infection.

OmpA Protein-Deficient Acinetobacter baumannii Outer Membrane Vesicles Trigger Reduced Inflammatory Response.

Multidrug resistant Acinetobacter baumannii shows a growing number of nosocomial infections worldwide during the last decade. The outer membrane vesicles (OMVs) produced by this bacterium draw increasing attention as a possible treatment target. OMVs have been implicated in the reduction of antibiotic level in the surrounding environment, transfer of virulence factors into the host cells, and induction of inflammatory response. Although the evidence on the involvement of OMVs in A. baumannii pathogenesis is currently growing, their role during inflammation is insufficiently explored. It is likely that bacteria, by secreting OMVs, can expand the area of their exposure and prepare surrounding matrix for infection. Here, we investigated the impact of A. baumannii OMVs on activation of macrophages in vitro. We show that OmpA protein present in A. baumannii OMVs substantially contributes to the proinflammatory response in J774 murine macrophages and to the cell death in both lung epithelium cells and macrophages. The loss of OmpA protein in OMVs, obtained from A. baumannii ∆ompA mutant, resulted in the altered expression of genes coding for IL-6, NLRP3 and IL-1ß proinflammatory molecules in macrophages in vitro. These results imply that OmpA protein in bacterial OMVs could trigger a more intense proinflammatory response.

The Toxin-Antitoxin Systems of the Opportunistic Pathogen Stenotrophomonas maltophilia of Environmental and Clinical Origin.

Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has recently emerged as a multidrug-resistant opportunistic pathogen causing bloodstream, respiratory, and urinary tract infections. The connection between the commensal environmental S. maltophilia and the opportunistic pathogen strains is still under investigation. Bacterial toxin-antitoxin (TA) systems have been previously associated with pathogenic traits, such as biofilm formation and resistance to antibiotics, which are important in clinical settings. The same species of the bacterium can possess various sets of TAs, possibly influencing their overall stress response. While the TA systems of other important opportunistic pathogens have been researched, nothing is known about the TA systems of S. maltophilia. Here, we report the identification and characterization of S. maltophilia type II TA systems and their prevalence in the isolates of clinical and environmental origins. We found 49 putative TA systems by bioinformatic analysis in S. maltophilia genomes. Despite their even spread in sequenced S. maltophilia genomes, we observed that relBE, hicAB, and previously undescribed COG3832-ArsR operons were present solely in clinical S. maltophilia isolates collected in Lithuania, while hipBA was more frequent in the environmental ones. The kill-rescue experiments in Escherichia coli proved higBA, hicAB, and relBE systems to be functional TA modules. Together with different TA profiles, the clinical S. maltophilia isolates exhibited stronger biofilm formation, increased antibiotic, and serum resistance compared to environmental isolates. Such tendencies suggest that certain TA systems could be used as indicators of virulence traits.

Capsule Protects Acinetobacter baumannii From Inter-Bacterial Competition Mediated by CdiA Toxin.

Currently, Acinetobacter baumannii is considered as one of the most important infectious agents causing hospital acquired infections worldwide. It has been observed that many clinically important pathogens express contact-dependent growth inhibition (CDI) phenomenon, which modulates cell-cell and cell-environment interactions, potentially allowing bacteria to adapt to ever-changing conditions. Mainly, these systems are used for the inhibition of the growth of genetically different individuals within the same species. In this work, by performing cell competition assays with three genotypically different (as determined by pulse-field gel electrophoresis) clinical A. baumannii isolates II-c, II-a, and II-a1, we show that A. baumannii capsule is the main feature protecting from CDI-mediated inhibition. We also observed that for one clinical isolate, the two-component BfmRS system, contributed to the resistance against CDI-mediated inhibition. Moreover, we were able to demonstrate, that the effector protein CdiA is released into the growth media and exhibits its inhibitory activity without the requirement of a cell-cell contact. Lastly, by evaluating the remaining number of the cells pre-mixed with the CdiA and performing live/dead assay, we demonstrate that purified CdiA protein causes a rapid cell growth arrest. Our results indicate, that capsule efficiently protects A. baumannii from a CDI-mediated inhibition by a clinical A. baumannii V15 strain, which is able to secrete CdiA effector into the growth media and cause target cell growth arrest without a cell-cell contact.

Microbial Diversity and Antimicrobial Resistance Profile in Microbiota From Soils of Conventional and Organic Farming Systems.

Soil is one of the biggest reservoirs of microbial diversity, yet the processes that define the community dynamics are not fully understood. Apart from soil management being vital for agricultural purposes, it is also considered a favorable environment for the evolution and development of antimicrobial resistance, which is due to its high complexity and ongoing competition between the microorganisms. Different approaches to agricultural production might have specific outcomes for soil microbial community composition and antibiotic resistance phenotype. Therefore in this study we aimed to compare the soil microbiota and its resistome in conventional and organic farming systems that are continually influenced by the different treatment (inorganic fertilizers and pesticides vs. organic manure and no chemical pest management). The comparison of the soil microbial communities revealed no major differences among the main phyla of bacteria between the two farming styles with similar soil structure and pH. Only small differences between the lower taxa could be observed indicating that the soil community is stable, with minor shifts in composition being able to handle the different styles of treatment and fertilization. It is still unclear what level of intensity can change microbial composition but current conventional farming in Central Europe demonstrates acceptable level of intensity for soil bacterial communities. When the resistome of the soils was assessed by screening the total soil DNA for clinically relevant and soil-derived antibiotic resistance genes, a low variety of resistance determinants was detected (resistance to ß-lactams, aminoglycosides, tetracycline, erythromycin, and rifampicin) with no clear preference for the soil farming type. The same soil samples were also used to isolate antibiotic resistant cultivable bacteria, which were predominated by highly resistant isolates of Pseudomonas, Stenotrophomonas, Sphingobacterium and Chryseobacterium genera. The resistance of these isolates was largely dependent on the efflux mechanisms, the soil Pseudomonas spp. relying mostly on RND, while Stenotrophomonas spp. and Chryseobacterium spp. on RND and ABC transporters.

Surface-Related Features and Virulence Among Acinetobacter baumannii Clinical Isolates Belonging to International Clones I and II.

Acinetobacter baumannii currently represents one of the most important nosocomial infection agent due to its multidrug-resistance and a propensity for the epidemic spread. The A. baumannii strains belonging to the international clonal lineages I (IC I) and II (IC II) are associated with the hospital outbreaks and a high virulence. However, the intra and inter lineage-specific features of strains belonging to these most worldwide spread A. baumannii clones are not thoroughly explored. In this study we have investigated a set of cell surface-related features of A. baumannii IC I (n = 20) and IC II (n = 16) lineage strains, representing 30 distinct pulsed-field gel electrophoresis types in the collection of clinical isolates obtained in Lithuanian tertiary care hospitals. We show that A. baumannii IC II strains are non-motile, do not form pellicle and display distinct capsular polysaccharide profile compared with the IC I strains. Moreover, in contrast to the overall highly hydrophobic IC I strains, IC II strains showed a greater variation in cell surface hydrophobicity. Within the IC II lineage, hydrophilic strains demonstrated reduced ability to form biofilm and adhere to the abiotic surfaces, also possessed twofold thicker cell wall and exhibited higher resistance to desiccation. Furthermore, these strains showed increased adherence to the lung epithelial cells and were more virulent in nematode and mouse infection model compared with the hydrophobic IC II strains. According to the polymerase chain reaction-based locus-typing, the reduction in hydrophobicity of IC II strains was not capsule or lipooligosaccharide locus type-dependent. Hence, this study shows that the most widespread A. baumannii clonal lineages I and II markedly differ in the series of cell surface-related phenotypes including the considerable phenotypic diversification of IC II strains at the intra-lineage level. These findings suggest that the genotypically related A. baumannii strains might evolve the features which could provide an advantage at the specific conditions outside or within the host.