- Volume 156, Issue 11, 2010

Heptose-containing oligosaccharides (OSs) are found in the outer core of the lipopolysaccharide (LPS) of a subset of non-typable Haemophilus influenzae (NTHi) strains. Candidate genes for the addition of either l-glycero-d-manno-heptose (ld-Hep) or d-glycero-d-manno-heptose (dd-Hep) and subsequent hexose sugars to these OSs have been identified from the recently completed genome sequences available for NTHi strains. losA1/losB1 and losA2/losB2 are two sets of related genes in which losA has homology to genes encoding glycosyltransferases and losB to genes encoding heptosyltransferases. Each set of genes is variably present across NTHi strains and is located in a region of the genome with an alternative gene organization between strains that contributes to LPS heterogeneity. Dependent upon the strain background, the LPS phenotype, structure and serum resistance of strains mutated in these genes were altered when compared with the relevant parent strain. Our studies confirm that losB1 and losB2 usually encode dd-heptosyl- and ld-heptosyl transferases, respectively, and that losA1 and losA2 encode glycosyltransferases that play a role in OS extensions of NTHi LPS.

The pathogenic fungus Candida albicans is able to cover its most potent proinflammatory cell wall molecules, the β-glucans, underneath a dense mannan layer, so that the pathogen becomes partly invisible for immune cells such as phagocytes. As the C. albicans histidine kinases Chk1p, Cos1p and CaSln1p had been reported to be involved in virulence and cell wall biosynthesis, we investigated whether deletion of the respective genes influences the activity of phagocytes against C. albicans. We found that among all histidine kinase genes, CHK1 plays a prominent role in phagocyte activation. Uptake of the deletion mutant Δchk1 as well as the acidification of Δchk1-carrying phagosomes was significantly increased compared with the parental strain. These improved activities could be correlated with an enhanced accessibility of the mutant β-1,3-glucans for immunolabelling. In addition, any inhibition of β-1,3-glucan-mediated phagocytosis resulted in a reduced uptake of Δchk1, while ingestion of the parental strain was hardly affected. Moreover, deletion of CHK1 caused an enhanced release of interleukins 6 and 10, indicating a stronger activation of the β-1,3-glucan receptor dectin-1. In conclusion, the Chk1p protein is likely to be involved in masking β-1,3-glucans from immune recognition. As there are no homologues of fungal histidine kinases in mammals, Chk1p has to be considered as a promising target for new antifungal agents.

Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the clp gene regulator (ClgR) of Mycobacterium tuberculosis activates the transcription of at least ten genes, including four that encode protease systems (ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, ClpB and the chaperonin Rv3269). Thus, M. tuberculosis ClgR controls a larger network of protein homeostatic and regulatory systems than ClgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for M. tuberculosis to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as M. tuberculosis lacking ClgR is deficient in the ability to control phagosome pH 1 h post-phagocytosis.

Larvae of Galleria mellonella, the greater wax moth, provide an alternative infection model for many human pathogens as they are amenable to use at elevated incubation temperatures (37 °C). This study and a parallel study by Mukherjee et al. [Mukherjee, K., Altincicek, B., Hain, T., Domann, E., Vilcinskas, A. & Chakraborty, T. (2010). Appl Environ Microbiol 76, 310–317] establish this insect host as an appropriate model to investigate the pathogenesis of Listeria species. In this study we show that inoculation with Listeria monocytogenes initiates a dynamic infection in G. mellonella and that production of the cytolysin listeriolysin O (LLO) is necessary for toxicity and bacterial growth. Production of LLO by the non-pathogenic species Lactococcus lactis is sufficient to induce mortality in the insect model. We employed real-time bioluminescence imaging to examine the dynamics of listerial growth and virulence gene expression in the G. mellonella model. Analysis of lux promoter fusions demonstrated significant induction of virulence gene expression upon introduction of the pathogen into insects at both 30 and 37 °C. The host response to listerial infection was examined which demonstrated that haemocyte destruction accompanies L. monocytogenes pathogenesis and is preceded by activation of the phenoloxidase system. Furthermore, we demonstrate that Listeria innocua is pathogenic to G. mellonella through a persistence mechanism that implicates an alternative mechanism for pathogenicity in this model.

Dental plaque formation is a developmental process involving cooperation and competition within a diverse microbial community, approximately 70 % of which is composed of an array of streptococci during the early stages of supragingival plaque formation. In this study, 79 cell-free culture supernatants from a variety of oral streptococci were screened to identify extracellular compounds that inhibit biofilm formation by the oral anaerobe Porphyromonas gingivalis strain 381. The majority of the streptococcal supernatants (61 isolates) resulted in lysis of P. gingivalis cells, and some (17 isolates) had no effect on cell viability, growth or biofilm formation. One strain, however, produced a supernatant that abolished biofilm formation without affecting growth rate. Analysis of this activity led to the discovery that a 48 kDa protein was responsible for the inhibition. Protein sequence identification and enzyme activity assays identified the effector protein as an arginine deiminase. To identify the mechanism(s) by which this protein inhibits biofilm formation, we began by examining the expression levels of genes encoding fimbrial subunits; surface structures known to be involved in biofilm development. Quantitative RT-PCR analysis revealed that exposure of P. gingivalis cells to this protein for 1 h resulted in the downregulation of genes encoding proteins that are the major subunits of two distinct types of thin, single-stranded fimbriae (fimA and mfa1). Furthermore, this downregulation occurred in the absence of arginine deiminase enzymic activity. Hence, our data indicate that P. gingivalis can sense this extracellular protein, produced by an oral streptococcus (Streptococcus intermedius), and respond by downregulating expression of cell-surface appendages required for attachment and biofilm development.

Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-14C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM.