Miguel A. Valvano

Antimicrobial resistance (AMR) is a global threat to human health and prosperity. Antimicrobial resistance breakers (ARBs), molecules that reverse AMR e.g., by blocking enzymes involved in bacterial membrane remodelling, are an attractive solution to restore the efficacy of existing antibiotics. We have identified several hits from in silico screening that restore polymyxin B (PmB) efficacy at concentrations of 250-500 µM in the clinically relevant ESKAPE pathogen Enterobacter bugandensis. The compounds act in tandem with PmB, but not with other antibiotics. They have no significant antibacterial effect themselves and may therefore be regarded as true resistance breakers. Preliminary results suggest that at least two of the three hit compounds may act via covalent bond formation with a target enzyme.

Exported polysaccharides play crucial functions in bacteria. Polysaccharide biosynthesis in the ubiquitous Wzx/Wzy- and ABC-transporter-dependent pathways starts with the transfer of a sugar-1-phosphate from a nucleotide-sugar donor to undecaprenyl phosphate, a reaction catalyzed by a phosphoglycosyl transferase (PGT). Both reaction substrates are limited and shared among multiple glycoconjugate pathways, raising the question of how bacteria regulate these pathways. In Myxococcus xanthu s, EpsZ, which belongs to the family of large monotopic PGTs (monoPGTs), starts the Wzx/Wzy-dependent exopolysaccharide (EPS) biosynthesis. The Dif chemosensory system regulates EPS biosynthesis by an unknown mechanism via the phosphorylated single-domain response regulator EpsW (EpsW~P). Here, we show that EpsW~P stimulates EPS biosynthesis at the post-translational level. Moreover, MiniTurbo-based proximity labeling experiments suggest that EpsW~P interacts directly with EpsZ. Additionally, heterologous expression of these two proteins in Salmonella enterica demonstrates that EpsW stimulates EpsZ enzymatic activity. S. enterica WbaP, the prototype large monoPGT, forms a functional homodimer, with dimerization involving a distinct cytoplasmic β-hairpin. However, AlphaFold-based structural modeling shows that EpsZ lacks this β-hairpin, suggesting an alternative mechanism for dimerization. Structural modeling of an EpsZ-EpsW heterocomplex suggests that EpsW~P, by direct interaction, promotes the formation of the stable EpsZ dimer. These findings suggest a new model for the regulation of polysaccharide biosynthesis in which EpsW~P allosterically facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating EPS biosynthesis at its initial step. Genomics and structural modeling suggest that the regulation of large monoPGTs by a single-domain response regulator is widespread in myxobacteria and potentially beyond. IMPORTANCE: Bacteria produce various polysaccharides with important biological functions and biotechnological applications. Polysaccharide synthesis is energy-costly and requires substrates that are in limited supply, raising the question of how bacteria regulate these pathways. Here, we explored the regulation of exopolysaccharide biosynthesis in Myxococcus xanthus . We demonstrate that the phosphorylated single-domain response regulator EpsW activates exopolysaccharide biosynthesis at the post-translational level by stimulating the activity of the phosphoglycosyl transferase EpsZ. By interacting with EpsZ, phosphorylated EpsW facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating exopolysaccharide biosynthesis at its initial step. We propose that this previously unrecognized regulatory mechanism is broadly conserved, not only in myxobacteria but also beyond.

Pseudomonas aeruginosa is an opportunistic pathogen frequently infecting the lower airways of individuals with cystic fibrosis (iwCF). We investigated colistin (CST) resistance mechanisms in P. aeruginosa ST3351 (Psa-ST3351) and their impact on bacterial virulence. Psa-ST3351, a novel CST-resistant sequence type, was isolated from a child with CF. We demonstrate that CST resistance in Psa-ST3351 depends on the addition of 4-amino-4-deoxy-L-arabinose to lipid A. Moreover, exposure to CST induced a non-mucoid-to-mucoid phenotypic switch characterized by enhanced swarming motility, decreased susceptibility to aminoglycosides, impaired ExoS secretion via the type-III secretion system, and increased bacterial competition, all hallmarks of a chronic infection phenotype. CST-treated Psa-ST3351 also exhibited reduced phagocytosis by macrophages, despite the induction of pro-inflammatory cytokines remaining unaffected, and enhanced the pathogenicity in a mouse pneumonia model. These findings suggest that CST-induced mucoid conversion in CST-resistant Psa-ST3351 May contribute to disease progression in iwCF, particularly when CST resistance and mucoid conversion remain undetected during CST nebulized therapy.

We have built PLASMIDITY, an automated pipeline to assemble short reads and characterize contigs searching for biologically relevant features for plasmid sequences detection applying machine learning methods, including contig length, multiplicity, circularity, insertion sequence composition, chromosome markers (rRNA, tRNA), plasmid markers (OriV, Rep, Mob/Rlx), G+C% variation, similarity with plasmid references, and k-mer profile. Using simulated WGS reads from 200 taxonomically diverse samples, we trained two Gradient Boosting classifiers, achieving a maximum F1 score of 0.901 in the test dataset. The comparison of the models against other plasmid prediction tools shows an improvement in the outcomes.

Diabetes mellitus is marked by chronic low-grade inflammation, increasing the risk of infections and vascular complications. In this study, we uncover a novel link between bacterial infection and epigenetic regulation in diabetes. We found that infection with Escherichia coli strongly induces the expression of histone deacetylase 11 (HDAC11) in endothelial cell-derived induced pluripotent stem cells (iPS-ECs) from diabetic donors. This upregulation of HDAC11 correlated with a surge in pro-inflammatory cytokine production and marked endothelial dysfunction. Strikingly, treatment with an HDAC11-specific inhibitor significantly dampened inflammatory signaling and restored endothelial function, pointing to a potential therapeutic strategy. Further mechanistic insights revealed a direct interaction between HDAC11 and STAT3, a key regulator of inflammatory gene expression. These findings position HDAC11 as a critical mediator of infection-induced vascular inflammation in diabetes and highlight it as a promising epigenetic target to combat endothelial damage and improve vascular health in diabetic patients.

Confocal microscopy provides information on host–pathogen interactions. To examine infection at the respiratory epithelial barrier, primary bronchial cells differentiated at an air-liquid interface (ALI) can be imaged and reconstructed as a 3D model. In this chapter, we describe a method for immunostaining and fluorescence imaging of well-differentiated bronchial epithelial tissue that has been exposed to the cystic fibrosis respiratory pathogen Achromobacter xylosoxidans.

Bacterial adaptation to stress can lead to phenotypic variants with diverse levels of niche competitiveness, pathogenicity, and antimicrobial resistance. In this work, we employed experimental evolution to investigate whether exposure to various stress conditions results in new phenotypic and metabolic properties in clinical and environmental strains of Vibrio cholerae . Our findings revealed the emergence of variants with metabolic and genetic variations and enhanced survival under stress compared to the parental isolates. Phenotypic changes in the evolved variants included colony morphology, biofilm formation, and the appearance of proteolytic and hemolytic activities. The variants demonstrated metabolic changes in the preferred use of carbon, nitrogen, phosphorous, and sulfur substrates, while the genetic changes included single nucleotide polymorphisms (SNPs), breakpoints, translocations, and single nucleotide insertions and deletions. Mutations in genes encoding EAL and HD-GYP domain-containing proteins correlated with increased biofilm formation and different colony morphotypes. The combined analysis of the metabolic and genomic data pointed to pathways implicated in stress survival. The environmental strains were generally more pathogenic than the clinical strains in the Galleria mellonella infection model prior to the experimental evolution, and these differences did not change in the evolved variants. This study highlights the contribution of stress conditions as drivers for the evolution of genetic modifications and metabolic adaptation in V. cholerae , which may explain the continuous evolution of El-Tor biotype strains toward variants with improved survival in the environment. IMPORTANCE: How Vibrio cholerae , the causative agent of cholera, survives during the periods between outbreaks remains a critical question. Using experimental evolution based on serial bacterial passages in culture media mimicking diverse environmental stress conditions, we investigated whether clinical and environmental isolates of V. cholerae develop changes in survival and in their metabolism. The evolved variants exhibited alterations in colony morphology, biofilm formation, and metabolism, including changes in the preferred use of carbon, nitrogen, phosphorous, and sulfur substrates. These changes were accompanied by various genetic modifications, notably in genes encoding second messenger molecules that regulate multiple biochemical pathways implicated in stress survival and increased pathogenic potential. Our results suggest a continuous evolution of V. cholerae strains toward variants displaying increased survival under environmental stress conditions that may also be encountered in the human host.

Background: Enterobacter species are opportunistic, multidrug resistant gram-negative bacteria associated with morbidity and mortality worldwide. Because very little is known about the infection biology of Enterobacter spp, we investigated the intracellular trafficking of a subset of Enterobacter clinical isolates, including colistin-resistant strains, within human macrophages and determined the macrophage response to the intracellular infection. Methods: Phagocytosis of 11 clinical isolates representing Enterobacter cloacae, Enterobacter bugandensis, Enterobacter kobei, Enterobacter xiangfangensis, Enterobacter roggenkampii, Enterobacter hoffmannii, and Enterobacter ludwigii was investigated in primary human macrophages. Intracellular bacterial trafficking was followed by confocal fluorescence microscopy, intracellular bacterial replication was assessed by bacterial enumeration, and a fluorescence dilution approach was used to follow bacterial cell division over time. Macrophage cell cytotoxicity was investigated by quantifying the release of lactate dehydrogenase during infection and by determining cleavage of the proinflammatory markers caspase-1, gasdermin D, and prointerleukin-1β. Results: Enterobacter isolates did not replicate in human macrophages, exhibiting long-term survival (up to 44 hours) within a modified late phagolysosome compartment. Survival did not correlate with colistin resistance, lipopolysaccharide modifications, or bacterial pathogenicity in the Galleria mellonella infection model. Intracellular bacteria induced low levels of macrophage cytotoxicity that correlated with absence of cleavage of proinflammatory markers in infected macrophages. Conclusions: Enterobacter spp clinical isolates can persist without replication inside human macrophages with minimal effects on cell integrity and inflammation. These observations could have implications for clinical outcome of patients that cannot readily clear Enterobacter infections, which can potentially lead to prolonged intracellular survival and infection relapse.

Summary: Background: The Enterobacter cloacae complex (Ecc) encompasses opportunistic Gram-negative bacteria demonstrating considerable phenotypic and genotypic diversity. Bloodstream, respiratory and urinary tract infections by Ecc bacteria are associated with morbidity and mortality worldwide. These infections are often difficult to treat since Ecc bacteria are resistant to penicillins, quinolones, aminoglycosides, and third-generation cephalosporins. Resistance also extends to carbapenems, leaving only polymyxins, such as colistin, as a last resort antibiotic for treatment. However, colistin resistance in Ecc isolates is also unexpectedly frequent. Despite extensive information on antibiotic resistance by Enterobacter species, much less is known about their infection biology. There are few reports on the survival and persistence of selected Enterobacter species in macrophages and epithelial cells, but how Enterobacter isolates interact with innate immune host cells upon engulfment remains unexplored. In this study, we have investigated the intracellular trafficking of a subset of antimicrobial resistant Ecc clinical isolates, including colistin-resistant strains, within human macrophages, and determined the macrophage response to the intracellular infection. Methods: Phagocytosis of 11 clinical Ecc isolates, including E. cloacae, E. bugandensis, E. kobei, E. xiangfangensis, E. roggenkampii, E. hoffmannii , and E. ludwigii was investigated in THP-1 and human monocyte derived macrophages (HMDMs). Confocal fluorescence microscopy was used to ascertain intracellular trafficking via co-localisation of cell markers with fluorescent bacteria. Intracellular bacterial replication was assessed by bacterial enumeration in cell lysates after killing extracellular bacteria and by a fluorescence dilution approach to follow the synthesis of the bacterial cell wall over time. Macrophage cell cytotoxicity was investigated by quantifying the release of lactate dehydrogenase during infection with all isolates. Two prototypic isolates, the E. cloacae ATCC13047 type strain and the E. bugandensis 104107, were used to explore in more detail the response of macrophages to the intracellular infection by determining cleavage of the proinflammatory markers caspase-1, gasdermin D and pro-interleukin-1β. Findings: We found that Ecc isolates do not replicate in human macrophages but survive within a modified late phagolysosome compartment. Survival occurred in all species investigated and did not correlate with colistin resistance, lipopolysaccharide modifications, or bacterial pathogenicity in the Galleria mellonella infection model. All isolates induced macrophage cell cytotoxicity at significantly lower levels than controls treated with lipopolysaccharide and nigericin treatment (to induce a proinflammatory response). Low cytotoxicity also correlated with absence of cleavage of proinflammatory markers in infected macrophages. Interpretation: Ecc species can survive without replication inside human macrophages with minimal effects on cell viability and inflammation. These observations could have implications in the clinical outcome of patients that cannot readily clear infecting Ecc bacteria. This can potentially lead to prolonged intracellular survival and infection relapse. Funding: Biotechnology and Biological Sciences Research Council grants BB/T005807/1 and BB/S006281/1. Research In context: Evidence before this study: We searched PubMed with the terms “Enterobacter” AND “macrophage”, “Enterobacter” AND “monocytes”, and “Enterobacter” AND “intracellular” for original articles published in English up to July 9, 2024. The search excluded terms “sakazakii” and “aerogenes” since E. aerogenes and E. sakazakii have been moved to the genus Klebsiella and Cronobacter , respectively. Of the 55, 15, and 181 studies we identified, respectively, only one reported testing Enterobacter cloacae phagocytosis. Another study reported intracellular bacterial communities in uroepithelial cells, which serve as a reservoir during urinary tract infection. One other study reported the isolation of E. hormaechei from human atherosclerotic tissue and described studies in THP-1 monocytic macrophages. A few earlier studies also reported Enterobacter cytotoxins affecting immune cells, and an E. cloacae polysaccharide capable of inducing apoptosis in epithelial cells. These studies did not investigate mechanisms and there have been no more recent follow-ups; importantly, it remains unclear if the strains employed in these studies were properly identified as Enterobacter species. Therefore, despite isolated observations of survival of Enterobacter isolates surviving in immune and epithelial cells, there is an overall knowledge gap in our understanding of these pathogens concerning intracellular survival compartments, kinetics of survival, and induction of macrophage cytotoxicity and inflammatory responses. Added value of this study: Our study is the first to investigate in detail how clinical isolates of various Enterobacter species can survive intracellularly in human macrophages. All isolates display multidrug antimicrobial resistance, including some with colistin resistance, and can survive intracellularly in human macrophages. Our data demonstrate that intracellular Enterobacter resides in vacuoles for up to 44 hours without replication. Colocalization experiments with various fluid phase and membrane cellular markers revealed that bacteria-containing vacuoles are modified late phagolysosomes, which do not accumulate the autophagosome marker LC3B. Intracellular bacterial survival did not associate to any Enterobacter species tested, the presence of colistin resistance, lipopolysaccharide modifications, or virulence in the Galleria mellonella infection model. Moreover, intracellular infection caused minimal cytotoxicity in macrophages without evidence of macrophage proinflammatory cell death by pyroptosis. Implications of all the available evidence: Our findings underscore the capacity of Enterobacter species, traditionally viewed as extracellular bacteria, to hideout in macrophages without inducing a significant inflammatory response. These properties may further complicate the treatment of antibiotic-resistant Enterobacter infections in susceptible populations such as the elderly and neonates. These findings open a door to the development of host-directed therapeutics to enhance bacterial clearance by macrophage-mediated killing.