- Volume 161, Issue 6, 2015

Helicobacter pylori is a major aetiological agent in the development of various gastroduodenal diseases. Its persistence in gastric mucosa is determined by the interaction between various host, microbial and environmental factors. The bacterium colonizes the gastric epithelium and induces activation of various chemokine mediators, including NFκB, the master regulator of inflammation. H. pylori infection is also associated with an increase in expression of cell cycle regulators, thereby leading to mucosal cell hyper-proliferation. Thus, H. pylori-associated infections manifest activation of key host response events, which inadvertently could lead to the establishment of chronic infection and neoplastic progression. This article reviews and elaborates the current knowledge in H. pylori-induced activation of various host signalling pathways that could promote cancer development. Special focus is placed on the inflammatory and proliferative responses that could serve as suitable biomarkers of infection, since a sustained cell proliferation in an environment rich in inflammatory cells is characteristic in H. pylori-associated gastric malignancies. Here, the role of ERK and WNT signalling in H. pylori-induced activation of inflammatory and proliferative responses respectively is discussed in detail. An in depth analysis of the underlying signalling pathways and interacting partners causing alterations in these crucial host responses could contribute to the development of successful therapeutic strategies for the prevention, management and treatment of H. pylori infection.

It is increasingly clear that rapid evolutionary dynamics are an important process in microbial ecology. Experimental evolution, wherein microbial evolution is observed in real-time, has revealed many instances of appreciable evolutionary change occurring on very short timescales of a few days or weeks in response to a variety of biotic and abiotic selection pressures. From clinical infections, including the chronic bacterial lung infections associated with cystic fibrosis that form a focus of my research, there is now abundant evidence suggesting that rapid evolution by infecting microbes contributes to host adaptation, treatment failure and worsening patient prognosis. However, disentangling the drivers of natural selection in complex infection environments is extremely challenging and limits our understanding of the selective pressures acting upon microbes in infections. Controlled evolution experiments can make a vital contribution to this by determining the causal links between predicted drivers of natural selection and the evolutionary responses of microbes. Integration of experimental evolution into studies of clinical infections is a key next step towards a better understanding of the causes and consequences of rapid microbial evolution in infections, and discovering how these evolutionary processes might be influenced to improve patient health.

A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2015, can be viewed via this link: Michael A. Brockhurst https://www.youtube.com/watch?v=N1bodVSl27E

The newly described species Haemophilus sputorum has been cultured from the upper respiratory tract of humans and appears to have little pathogenic potential. The species encodes a capsular biosynthesis locus of approximately 12 kb composed of three distinct regions. Region I and III genes, involved in export and processing of the capsular material, show high similarity to the corresponding genes in capsulate lineages of the pathogenic species Haemophilus influenzae; indeed, standard bexA and bexB PCRs for detection of capsulated strains of H. influenzae give positive results with strains of H. sputorum. Three ORFs are present in region II of the sequenced strain of H. sputorum, of which a putative phosphotransferase showed homology with corresponding genes from H. influenzae serotype c and f. Phylogenetic analysis of housekeeping genes from 24 Pasteurellaceae species showed that H. sputorum was only distantly related to H. influenzae. In contrast to H. influenzae, the capsule locus in H. sputorum is not associated with transposases or other transposable elements. Our data suggest that the capsule locus of capsulate lineages of H. influenzae may have been recruited relatively recently from the commensal species H. sputorum by horizontal gene transfer.

The Yersinia pestis PhoPQ gene regulatory system is induced during infection of the flea digestive tract and is required to produce adherent biofilm in the foregut, which greatly enhances bacterial transmission during a flea bite. To understand the in vivo context of PhoPQ induction and to determine PhoP-regulated targets in the flea, we undertook whole-genome comparative transcriptional profiling of Y. pestis WT and ΔphoP strains isolated from infected fleas and from temperature-matched in vitro planktonic and flow-cell biofilm cultures. In the absence of PhoP regulation, the gene expression program indicated that the bacteria experienced diverse physiological stresses and were in a metabolically less active state. Multiple stress response genes, including several toxin–antitoxin loci and YhcN family genes responsible for increased acid tolerance, were upregulated in the phoP mutant during flea infection. The data implied that PhoPQ was induced by low pH in the flea gut, and that PhoP modulated physiological adaptation to acid and other stresses encountered during infection of the flea. This adaptive response, together with PhoP-dependent modification of the bacterial outer surface that includes repression of pH 6 antigen fimbriae, supports stable biofilm development in the flea foregut.

From a tomb in Upper Egypt we isolated a strain of Penicillium chrysogenum that was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some micro-organisms in adverse environments. We tested type strains of Pe . chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. Fourier transform IR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of Pe. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription.

Aspartate and glutamate are two key amino acids used in biosynthesis of many amino acids that play vital role in cellular metabolism. Aspartate aminotransferases (AspATs) are required for channelling nitrogen (N2) between Glu and Asp in all life forms. Biochemical and genetic characterization of AspATs have been lacking in N2-fixing cyanobacteria. In this report, five putative AspAT genes (alr1039, all2340, alr2765, all4327 and alr4853) were identified in the N2-fixing heterocystous cyanobacterium Anabaena sp. PCC 7120. Five recombinant C-terminal hexahistidine-tagged AspATs (AspAT-H6) were overexpressed in Escherichia coli and purified to homogeneity. Biochemical analysis demonstrated that these five putative AspATs have authentic AspAT activity in vitro using aspartate as an amino donor. However, the enzymic activities of the five AspATs differed in vitro. Alr4853-H6 showed the highest AspAT activity, while the enzymic activity for the other four AspATs ranged from 6.5 to 53.7 % activity compared to Alr4853 (100 %). Genetic characterization of the five AspAT genes was also performed by inactivating each individual gene. All of the five AspAT knockout mutants exhibited reduced diazotrophic growth, and alr4853 was further identified to be a Fox gene (requiring fixed N2 for growth in the presence of oxygen). Four out of five P aspAT -gfp transcriptional fusions were constitutively expressed in both diazotrophic and nitrate-dependent growth conditions. Quantitative reverse transcriptase PCR showed that alr4853 expression was increased by 2.3-fold after 24 h of N2 deprivation. Taken together, these findings add to our understanding of the role of AspATs in N2-fixing within heterocystous cyanobacteria.

The resuscitation-promoting factors of Mycobacterium tuberculosis are hydrolytic enzymes, which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling enzyme similar to the lytic transglycosylase of Escherichia coli, is required for reactivation of M. tuberculosis from chronic infection in vivo, underscoring the need to understand its transcriptional regulation. Here, we identified the transcriptional and translational start points of rpfB, and suggested from rpf promoter-driven GFP expression and in vitro transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrated that rpfB transcription is regulated by MtrA – the response regulator of the essential two-component system MtrAB. Association of MtrA with the rpfB promoter region in vivo was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down from lysates of M. tuberculosis using a biotinylated DNA fragment encompassing the MtrA-binding site on the rpfB promoter, confirming that MtrA binds to the rpfB promoter. Enhanced GFP fluorescence driven by the rpfB promoter, upon deletion of the MtrA-binding site, and repression of rpfB expression, upon overexpression of MtrA, suggested that MtrA functions as a repressor of rpfB transcription. This was corroborated by EMSAs showing diminished association of RNA polymerase (RNAP) with the rpfB promoter in the presence of MtrA. In vitro transcription assays confirmed that MtrA inhibits RNAP-driven rpfB transcription.