Using signature-tagged transposon mutagenesis, we isolated 23 Mycobacterium tuberculosis mutants, corresponding to 21 genes or genetic regions, attenuated in their ability to parasitize human macrophages. Mutants disrupted in the ABC transporter-encoding genes Rv0986 and Rv0987 were further characterized as being impaired in their ability to bind to host cells.

Glycosylated p-hydroxybenzoic acid methyl esters and structurally related phenolphthiocerol glycolipids are important virulence factors of Mycobacterium tuberculosis. Although both types of molecules are thought to be derived from p-hydroxybenzoic acid, the origin of this putative biosynthetic precursor in mycobacteria remained to be established. We describe the characterization of a transposon mutant of M. tuberculosis deficient in the production of all forms of p-hydroxybenzoic acid derivatives. The transposon was found to be inserted in Rv2949c, a gene located in the vicinity of the polyketide synthase gene pks15/1, involved in the elongation of p-hydroxybenzoate to phenolphthiocerol in phenolic glycolipid-producing strains. A recombinant form of the Rv2949c enzyme was produced in the fast-growing non-pathogenic Mycobacterium smegmatis and purified to near homogeneity. The recombinant enzyme catalyzed the removal of the pyruvyl moiety of chorismate to form p-hydroxybenzoate with an apparent K_m value for chorismate of 19.7 μm and a k_cat value of 0.102 s^-1. Strong inhibition of the reaction by p-hydroxybenzoate but not by pyruvate was observed. These results establish Rv2949c as a chorismate pyruvate-lyase responsible for the direct conversion of chorismate to p-hydroxybenzoate and identify Rv2949c as the sole enzymatic source of p-hydroxybenzoic acid in M. tuberculosis.

The locus lex2, comprising lex2A and lex2B, contributes to the phase-variable expression of lipopolysaccharide (LPS) of Haemophilus influenzae and was found to be present in 74 % of strains investigated. lex2A contains 5′-GCAA repeats which vary in number from 4 to 46 copies between strains. The locus was cloned from the serotype b strains RM7004 and RM153 and showed >99 % nucleotide sequence identity between these strains and the published lex2 sequence. Disruption of the lex2B gene in strain RM7004 resulted in truncation of some LPS glycoforms, shown by gel fractionation, with only one glycoform reacting with a digalactoside-specific monoclonal antibody, 4C4, compared with four LPS glycoforms in the more elongated LPS of the parent strain. Mass spectrometry and NMR analyses of LPS from the lex2B mutant revealed loss of the terminal digalactoside as well as the second β-glucose extending from the first heptose of the inner core. The authors conclude that Lex2B is the β-(1-4)-glucosyltransferase that adds the second β-glucose to the first β-glucose as part of the oligosaccharide extension from the first heptose of the LPS of strain RM7004. Investigation of the expression of the lex2 locus indicated that the genes are co-transcribed and that both reading frames are required for addition of this second β-glucose in a phase-variable manner.

Fecal microbiota transplantation (FMT) is highly effective in recurrent Clostridioides difficile infection (CDI); increasing evidence supports FMT in severe or fulminant Clostridioides difficile infection (SFCDI). However, the multifactorial mechanisms that underpin the efficacy of FMT are not fully understood. Systems biology approaches using high-throughput technologies may help with mechanistic dissection of host-microbial interactions. Here, we have undertaken a deep phenomics study on four adults receiving sequential FMT for SFCDI, in which we performed a longitudinal, integrative analysis of multiple host factors and intestinal microbiome changes. Stool samples were profiled for changes in gut microbiota and metabolites and blood samples for alterations in targeted epigenomic, metabonomic, glycomic, immune proteomic, immunophenotyping, immune functional assays, and T-cell receptor (TCR) repertoires, respectively. We characterised temporal trajectories in gut microbial and host immunometabolic data sets in three responders and one non-responder to sequential FMT. A total of 562 features were used for analysis, of which 78 features were identified, which differed between the responders and the non-responder. The observed dynamic phenotypic changes may potentially suggest immunosenescent signals in the non-responder and may help to underpin the mechanisms accompanying successful FMT, although our study is limited by a small sample size and significant heterogeneity in patient baseline characteristics. Our multi-omics integrative longitudinal analytical approach extends the knowledge regarding mechanisms of efficacy of FMT and highlights preliminary novel signatures, which should be validated in larger studies.