Drug efflux and lipid A modification by 4-L-aminoarabinose are key mechanisms of polymyxin B resistance in the sepsis pathogen Enterobacter bugandensis.

OBJECTIVES: A concern with the ESKAPE pathogen, Enterobacter bugandensis, and other species of the Enterobacter cloacae complex, is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins. METHODS: Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis. RESULTS: In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain, W3110. The efflux pump, AcrABTolC, and the two component regulators, PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modelling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps. CONCLUSION: Our results support a model where drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.

Phage treatment of Pseudomonas aeruginosa yields a phage-resistant population with different susceptibility to innate immune responses and mild effects on metabolic profiles.

In this study, we have investigated innate immune activation capacity and metabolic features of a population of P. aeruginosa PAO1 phage-resistant mutants with diverse genetic modification (large genomic deletions and point mutations) arising after exposure to phages targetting lipopolysaccharide (LPS) or Type-4 pili (T4P). Deletions led to the loss of genes involved in LPS synthesis, cell envelope permeability, efflux systems, biofilm production, oxidative stress tolerance, and DNA repair. Loss of LPS O antigen resulted in bacterial sensitivity to serum complement and stimulation of inflammatory cascades but did not cause increased phagocytosis, while T4P phage-resistant mutants were more effectively phagocytized than LPS-defective mutants. Changes in the utilization of different carbon, nitrogen, sulphur, and phosphorus sources were identified, especially in mutants where the two phage DNA persisted in the bacterial population (pseudolysogeny). However, the metabolic changes did not directly correlate with single-gene mutations or the large gene deletions, suggesting they reflect adaptive changes to the gene modifications that arise during the selection of resistant mutants. In contrast, phage-resistant mutants were susceptible to humoral innate immune responses, suggesting that phage resistance may be a beneficial outcome of phage therapy.

Distribution and diversity of type VI secretion system clusters in Enterobacter bugandensis and Enterobacter cloacae.

Gram-negative bacteria use type VI secretion systems (T6SSs) to antagonize neighbouring cells. Although primarily involved in bacterial competition, the T6SS is also implicated in pathogenesis, biofilm formation and ion scavenging. Enterobacter species belong to the ESKAPE pathogens, and while their antibiotic resistance has been well studied, less is known about their pathogenesis. Here, we investigated the distribution and diversity of T6SS components in isolates of two clinically relevant Enterobacter species, E. cloacae and E. bugandensis. T6SS clusters are grouped into four types (T6SSi-T6SSiv), of which type i can be further divided into six subtypes (i1, i2, i3, i4a, i4b, i5). Analysis of a curated dataset of 31 strains demonstrated that most of them encode T6SS clusters belonging to the T6SSi type. All T6SS-positive strains possessed a conserved i3 cluster, and many harboured one or two additional i2 clusters. These clusters were less conserved, and some strains displayed evidence of deletion. We focused on a pathogenic E. bugandensis clinical isolate for comprehensive in silico effector prediction, with comparative analyses across the 31 isolates. Several new effector candidates were identified, including an evolved VgrG with a metallopeptidase domain and a Tse6-like protein. Additional effectors included an anti-eukaryotic catalase (KatN), M23 peptidase, PAAR and VgrG proteins. Our findings highlight the diversity of Enterobacter T6SSs and reveal new putative effectors that may be important for the interaction of these species with neighbouring cells and their environment.

The Achromobacter type 3 secretion system drives pyroptosis and immunopathology via independent activation of NLRC4 and NLRP3 inflammasomes.

How the opportunistic Gram-negative pathogens of the genus Achromobacter interact with the innate immune system is poorly understood. Using three Achromobacter clinical isolates from two species, we show that the type 3 secretion system (T3SS) is required to induce cell death in human macrophages by inflammasome-dependent pyroptosis. Macrophages deficient in the inflammasome sensors NLRC4 or NLRP3 undergo pyroptosis upon bacterial internalization, but those deficient in both NLRC4 and NLRP3 do not, suggesting either sensor mediates pyroptosis in a T3SS-dependent manner. Detailed analysis of the intracellular trafficking of one isolate indicates that the intracellular bacteria reside in a late phagolysosome. Using an intranasal mouse infection model, we observe that Achromobacter damages lung structure and causes severe illness, contingent on a functional T3SS. Together, we demonstrate that Achromobacter species can survive phagocytosis by promoting macrophage cell death and inflammation by redundant mechanisms of pyroptosis induction in a T3SS-dependent manner.

Polymyxin Resistance and Heteroresistance Are Common in Clinical Isolates of Achromobacter Species and Correlate with Modifications of the Lipid A Moiety of Lipopolysaccharide.

The Achromobacter genus includes opportunistic pathogens that can cause chronic infections in immunocompromised patients, especially in people with cystic fibrosis (CF). Treatment of Achromobacter infections is complicated by antimicrobial resistance. In this study, a collection of Achromobacter clinical isolates, from CF and non-CF sources, was investigated for polymyxin B (PmB) resistance. Additionally, the effect of PmB challenge in a subset of isolates was examined and the presence of PmB-resistant subpopulations within the isolates was described. Further, chemical and mass spectrometry analyses of the lipid A of Achromobacter clinical isolates enabled the determination of the most common structures and showed that PmB challenge was associated with lipid A modifications that included the addition of glucosamine and palmitoylation and the concomitant loss of the free phosphate at the C-1 position. This study demonstrates that lipid A modifications associated with PmB resistance are prevalent in Achromobacter and that subresistant populations displaying the addition of positively charged residues and additional acyl chains to lipid A can be selected for and isolated from PmB-sensitive Achromobacter clinical isolates. IMPORTANCE Achromobacter species can cause chronic and potentially severe infections in immunocompromised patients, especially in those with cystic fibrosis. Bacteria cannot be eradicated due to Achromobacter's intrinsic multidrug resistance. We report that intrinsic resistance to polymyxin B (PmB), a last-resort antimicrobial peptide used to treat infections by multiresistant bacteria, is prevalent in Achromobacter clinical isolates; many isolates also display increased resistance upon PmB challenge. Analysis of the lipopolysaccharide lipid A moiety of several Achromobacter species reveals a penta-acylated lipid A, which in the PmB-resistant isolates was modified by the incorporation of glucosamine residues, an additional acyl chain, loss of phosphates, and hydroxylation of acyl chains, all of which can enhance PmB resistance in other bacteria. We conclude that PmB resistance, particularly in Achromobacter isolates from chronic respiratory infections, is a common phenomenon, and that Achromobacter lipid A displays modifications that may confer increased resistance to polymyxins and potentially other antimicrobial peptides.

Bacterial conversion of a host weapon into a nutritional signal.

Bacteria engulfed by phagocytic cells must resist oxidation damage and adapt to cellular hypoxia, but the mechanisms involved in this process are not completely elucidated. Recent work by Kim et al. in the Journal of Biological Chemistry investigated how the intracellular pathogen Salmonella enterica activates gene expression required to counteract oxidative damage. The authors show that this bacterium utilizes host oxidative molecules to activate regulatory proteins that enhance the production of effector molecules, counteracting the host weapon NADPH oxidase and inducing a protective response.

Comparative analysis of Vibrio cholerae isolates from Ghana reveals variations in genome architecture and adaptation of outbreak and environmental strains.

Recurrent epidemics of cholera denote robust adaptive mechanisms of Vibrio cholerae for ecological shifting and persistence despite variable stress conditions. Tracking the evolution of pathobiological traits requires comparative genomic studies of isolates from endemic areas. Here, we investigated the genetic differentiation among V. cholerae clinical and environmental isolates by highlighting the genomic divergence associated with gene decay, genome plasticity, and the acquisition of virulence and adaptive traits. The clinical isolates showed high phylogenetic relatedness due to a higher frequency of shared orthologs and fewer gene variants in contrast to the evolutionarily divergent environmental strains. Divergence of the environmental isolates is linked to extensive genomic rearrangements in regions containing mobile genetic elements resulting in numerous breakpoints, relocations, and insertions coupled with the loss of virulence determinants acf, zot, tcp, and ctx in the genomic islands. Also, four isolates possessed the CRISPR-Cas systems with spacers specific for Vibrio phages and plasmids. Genome synteny and homology analysis of the CRISPR-Cas systems suggest horizontal acquisition. The marked differences in the distribution of other phage and plasmid defense systems such as Zorya, DdmABC, DdmDE, and type-I Restriction Modification systems among the isolates indicated a higher propensity for plasmid or phage disseminated traits in the environmental isolates. Our results reveal that V. cholerae strains undergo extensive genomic rearrangements coupled with gene acquisition, reflecting their adaptation during ecological shifts and pathogenicity.

Exploring the Topology of Cytoplasmic Membrane Proteins Involved in Lipopolysaccharide Biosynthesis by in Silico and Biochemical Analyses.

In the absence of a tri-dimensional structure, revealing the topology of a membrane protein provides relevant information to identify the number and orientation of transmembrane helices and the localization of critical amino acid residues, contributing to a better understanding of function and intermolecular associations. Topology can be predicted in silico by bioinformatic analysis or solved by biochemical methods. In this chapter, we describe a pipeline employing bioinformatic approaches for the prediction of membrane protein topology, followed by experimental validation through the substituted-cysteine accessibility method and the analysis of the protein's oligomerization state.

An evolutionary conserved detoxification system for membrane lipid-derived peroxyl radicals in Gram-negative bacteria.

How double-membraned Gram-negative bacteria overcome lipid peroxidation is virtually unknown. Bactericidal antibiotics and superoxide ion stress stimulate the transcription of the Burkholderia cenocepacia bcnA gene that encodes a secreted lipocalin. bcnA gene orthologs are conserved in bacteria and generally linked to a conserved upstream gene encoding a cytochrome b561 membrane protein (herein named lcoA, lipocalin-associated cytochrome oxidase gene). Mutants in bcnA, lcoA, and in a gene encoding a conserved cytoplasmic aldehyde reductase (peroxidative stress-associated aldehyde reductase gene, psrA) display enhanced membrane lipid peroxidation. Compared to wild type, the levels of the peroxidation biomarker malondialdehyde (MDA) increase in the mutants upon exposure to sublethal concentrations of the bactericidal antibiotics polymyxin B and norfloxacin. Microscopy with lipid peroxidation-sensitive fluorescent probes shows that lipid peroxyl radicals accumulate at the bacterial cell poles and septum and peroxidation is associated with a redistribution of anionic phospholipids and reduced antimicrobial resistance in the mutants. We conclude that BcnA, LcoA, and PsrA are components of an evolutionary conserved, hitherto unrecognized peroxidation detoxification system that protects the bacterial cell envelope from lipid peroxyl radicals.

Remodelling of the Gram-negative bacterial Kdo2-lipid A and its functional implications.

The lipopolysaccharide (LPS) is a characteristic molecule of the outer leaflet of the Gram-negative bacterial outer membrane, which consists of lipid A, core oligosaccharide, and O antigen. The lipid A is embedded in outer membrane and provides an efficient permeability barrier, which is particularly important to reduce the permeability of antibiotics, toxic cationic metals, and antimicrobial peptides. LPS, an important modulator of innate immune responses ranging from localized inflammation to disseminated sepsis, displays a high level of structural and functional heterogeneity, which arise due to regulated differences in the acylation of the lipid A and the incorporation of non-stoichiometric modifications in lipid A and the core oligosaccharide. This review focuses on the current mechanistic understanding of the synthesis and assembly of the lipid A molecule and its most salient non-stoichiometric modifications.

Protein with negative surface charge distribution, Bnr1, shows characteristics of a DNA-mimic protein and may be involved in the adaptation of Burkholderia cenocepacia.

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium with a large genome of ∼8 Mb that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. We previously identified an immunoreactive acidic protein encoded on replicon 3, BCAS0292. Deletion of the BCAS0292 gene significantly altered the abundance of 979 proteins by 1.5-fold or more; 19 proteins became undetectable while 545 proteins showed ≥1.5-fold reduced abundance, suggesting the BCAS0292 protein is a global regulator. Moreover, the ∆BCAS0292 mutant showed a range of pleiotropic effects: virulence and host-cell attachment were reduced, antibiotic susceptibility was altered, and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. In silico prediction of its three-dimensional structure revealed BCAS0292 presents a dimeric ß-structure with a negative surface charge. The ΔBCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. Three proteins were identified in pull-downs with FLAG-tagged BCAS0292, including the Histone H1-like protein, HctB, which is recognized as a global transcriptional regulator. We propose that BCAS0292 protein, which we have named Burkholderia negatively surface-charged regulatory protein 1 (Bnr1), acts as a DNA-mimic and binds to DNA-binding proteins, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.

Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of Action in Human Cells and In Vivo Activity.

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA.

Defining chaperone-usher fimbriae repertoire in Serratia marcescens.

Chaperone-usher (CU) fimbriae are surface organelles particularly prevalent among the Enterobacteriaceae. Mainly associated to their adhesive properties, CU fimbriae play key roles in biofilm formation and host cell interactions. Little is known about the fimbriome composition of the opportunistic human pathogen Serratia marcescens. Here, by using a search based on consensus fimbrial usher protein (FUP) sequences, we identified 421 FUPs across 39 S. marcescens genomes. Further analysis of the FUP-containing loci allowed us to classify them into 20 conserved CU operons, 6 of which form the S. marcescens core CU fimbriome. A new systematic nomenclature is proposed according to FUP sequence phylogeny. We also established an in vivo transcriptional assay comparing CU promoter expression between an environmental and a clinical isolate of S. marcescens, which revealed that promoters from 3 core CU operons (referred as fgov, fpo, and fps) are predominantly expressed in the two strains and might represent key core adhesion appendages contributing to S. marcescens pathogenesis.

A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for detection of glanders infections.

We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans, and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

Macrophage dysfunction in cystic fibrosis: Nature or nurture?

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect the homeostasis of chloride flux by epithelial cells. This has deleterious consequences, especially in respiratory epithelia, where the defect results in mucus accumulation distinctive of cystic fibrosis. CFTR is, however, also expressed in phagocytic cells, like macrophages. Immune cells are highly sensitive to conditioning by their environment; thus, CFTR dysfunction in epithelia influences macrophages by affecting the lung milieu, but the mutations also appear to be directly consequential for intrinsic macrophage functions. Particular mutations can alter CFTR's folding, traffic of the protein to the membrane and function. As such, understanding the intrinsic effects of CFTR mutation requires distinguishing the secondary effects of misfolded CFTR on cell stress pathways from the primary defect of CFTR dysfunction/absence. Investigations into CFTR's role in macrophages have exploited various models, each with their own advantages and limitations. This review summarizes these methodologic approaches, discussing their physiological correspondence and highlighting key findings. The controversy surrounding CFTR-dependent acidification is used as a case study to highlight difficulties in commensurability across model systems. Recent work in macrophage biology, including polarization and host-pathogen interaction studies, brought into the context of CFTR research, offers potential explanations for observed discrepancies between studies. Moreover, the rapid advancement of novel gene editing technologies and new macrophage model systems makes this assessment of the field's models and methodologies timely.

Current Advances in Burkholderia Vaccines Development.

The genus Burkholderia includes a wide range of Gram-negative bacterial species some of which are pathogenic to humans and other vertebrates. The most pathogenic species are Burkholderia mallei, Burkholderia pseudomallei, and the members of the Burkholderia cepacia complex (Bcc). B. mallei and B. pseudomallei, the cause of glanders and melioidosis, respectively, are considered potential bioweapons. The Bcc comprises a subset of Burkholderia species associated with respiratory infections in people with chronic granulomatous disease and cystic fibrosis. Antimicrobial treatment of Burkholderia infections is difficult due to the intrinsic multidrug antibiotic resistance of these bacteria; prophylactic vaccines provide an attractive alternative to counteract these infections. Although commercial vaccines against Burkholderia infections are still unavailable, substantial progress has been made over recent years in the development of vaccines against B. pseudomallei and B. mallei. This review critically discusses the current advances in vaccine development against B. mallei, B. pseudomallei, and the Bcc.

An Overview of Anti-Eukaryotic T6SS Effectors.

The type VI secretion system (T6SS) is a transmembrane multiprotein nanomachine employed by many Gram-negative bacterial species to translocate, in a contact-dependent manner, effector proteins into adjacent prokaryotic or eukaryotic cells. Typically, the T6SS gene cluster encodes at least 13 conserved core components for the apparatus assembly and other less conserved accessory proteins and effectors. It functions as a contractile tail machine comprising a TssB/C sheath and an expelled puncturing device consisting of an Hcp tube topped by a spike complex of VgrG and PAAR proteins. Contraction of the sheath propels the tube out of the bacterial cell into a target cell and leads to the injection of toxic proteins. Different bacteria use the T6SS for specific roles according to the niche and versatility of the organism. Effectors are present both as cargo (by non-covalent interactions with one of the core components) or specialized domains (fused to structural components). Although several anti-prokaryotic effectors T6SSs have been studied, recent studies have led to a substantial increase in the number of characterized anti-eukaryotic effectors. Against eukaryotic cells, the T6SS is involved in modifying and manipulating diverse cellular processes that allows bacteria to colonize, survive and disseminate, including adhesion modification, stimulating internalization, cytoskeletal rearrangements and evasion of host innate immune responses.

Complete Genome Sequence of Burkholderia cenocepacia K56-2, an Opportunistic Pathogen.

Burkholderia cenocepacia K56-2, an opportunistic bacterium for people with cystic fibrosis (CF), belongs to the Burkholderia cepacia complex (Bcc) and is consistently used as a model pathogen. We describe here the closed genome sequence for this strain, which will help advance research in B. cenocepacia biology and omics studies.