Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo.

The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem

Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe

Integrated analysis of patient networks and plasmid genomes reveals a regional, multi-species outbreak of carbapenemase-producing Enterobacterales carrying both blaIMP and mcr-9 genes

Background Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 across a London regional network. Methods We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE positive patients. Genomes of IMP-encoding CPE isolates were overlayed with patient contacts to imply potential transmission events. Results Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, E. coli); 86% (72/84) harboured an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68/72). Phylogenetic analysis of IncHI2 plasmids identified three lineages showing significant association with patient contacts and movements between four hospital sites and across medical specialities, which was missed on initial investigations. Conclusions Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multi-modal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks

High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt