- Volume 158, Issue 6, 2012

The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1β- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.

Parachlamydia acanthamoebae is an obligate intracellular bacterium that infects free-living amoebae (Acanthamoeba), and is a potential human pathogen associated with hospital-acquired pneumonia. The attachment mechanism of this bacteria to host cells is crucial in bacterial pathogenesis, yet remains undetermined. Hence, we obtained monoclonal antibodies (mAbs) specific to either P. acanthamoebae or amoebae in an attempt to elucidate the attachment mechanism involved. Hybridomas of 954 clones were assessed, and we found that four mAbs (mAb38, mAb300, mAb311, mAb562) that were reactive to the amoebae significantly inhibited bacterial attachment. All mAbs recognized amoebal released molecules, and mAb311 also recognized the amoebal surface. mAbs reacted with the bacteria not only within amoebae, but also when they were released from amoebae (except mAb311). Furthermore, a serine protease inhibitor had an inhibitory effect on the bacterial attachment to amoebae, although none of the mAbs had any synergistic effect on the inhibition of attachment by the protease inhibitor. Taken together, we conclude that concurrent P. acanthoamebae attachment to amoebae is required for several amoebal released molecules and serine protease activity, implying the existence of a complicated host–parasite relationship.

The capacity to form microscopic cords (cording) of Mycobacterium species has been related to their virulence. The compounds responsible for cording are unknown, but a recent study has shown that cording could be related to the fine structure of α-mycolic acids. This investigation attributes the need for a proximal cyclopropane in α-mycolic acids for cording in Mycobacterium tuberculosis and Mycobacterium bovis BCG and proposes cyclopropanases as good targets for new chemotherapeutic agents. As other Mycobacterium species in addition to M. tuberculosis and M. bovis form microscopic cords, it would be of major interest to know whether the relationship between proximal cyclopropanation of α-mycolic acids and cording could be extended to non-tuberculous mycobacteria. In this study, we have examined the correlation between the cording and cyclopropanation of α-mycolic acids in two species, Mycobacterium brumae and Mycobacterium fallax. Scanning electron microscopy images showed, for the first time to our knowledge, the fine structure of microscopic cords of M. brumae and M. fallax, confirming that these two species form true cords. Furthermore, NMR analysis performed on the same cording cultures corroborates the absence of cyclopropane rings in their α-mycolic acids. Therefore, we can conclude that the correlation between cording and cyclopropanation of α-mycolic acids cannot be extended to all mycobacteria. As M. brumae and M. fallax grow rapidly and have a simple pattern of mycolic acids (only α-unsaturated mycolic acids), we propose these two species as suitable models for the study of the role of mycolic acids in cording.

Certain heavy metal ions such as copper and zinc serve as essential cofactors of many enzymes, but are toxic at high concentrations. Thus, intracellular levels have to be subtly balanced. P-type ATPases of the PIB-subclass play a major role in metal homeostasis. The thermoacidophile Sulfolobus solfataricus possesses two PIB-ATPases named CopA and CopB. Both enzymes are present in cells grown in copper-depleted medium and are accumulated upon an increase in the external copper concentration. We studied the physiological roles of both ATPases by disrupting genes copA and copB. Neither of them affected the sensitivity of S. solfataricus to reactive oxygen species, nor were they a strict prerequisite to the biosynthesis of the copper protein cytochrome oxidase. Deletion mutant analysis demonstrated that CopA is an effective copper pump at low and high copper concentrations. CopB appeared to be a low-affinity copper export ATPase, which was only relevant if the media copper concentration was exceedingly high. CopA and CopB thus act as resistance factors to copper ions at overlapping concentrations. Moreover, growth tests on solid media indicated that both ATPases are involved in resistance to silver.

Genome sequence analysis of different fungi of the family Arthrodermataceae revealed the presence of a gene cluster consisting of five genes with high sequence similarity to those involved in the early common steps of ergot alkaloid biosynthesis in Aspergillus fumigatus and Claviceps purpurea. To provide evidence that this cluster is involved in ergot alkaloid biosynthesis, the gene ARB_04646 of the fungus Arthroderma benhamiae was cloned into pQE60 and expressed in Escherichia coli. Enzyme assays with the soluble tetrameric His6-tagged protein proved unequivocally that the deduced gene product, here termed ChaDH, catalysed the oxidation of chanoclavine-I in the presence of NAD+, resulting in the formation of chanoclavine-I aldehyde. The enzyme product was unequivocally proven by NMR and MS analyses. Therefore, ChaDH functions as a chanoclavine-I dehydrogenase. K m values for chanoclavine-I and NAD+ were 0.09 and 0.36 mM, respectively. Turnover number was 0.76 s–1.

Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c–cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K d value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of 63NiCl2. The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.

Proteins belonging to the PII family coordinate cellular nitrogen metabolism by direct interaction with a variety of enzymes, transcriptional regulators and transporters. The sensing function of PII relies on its ability to bind the nitrogen/carbon signalling molecule 2-oxoglutarate (2-OG). In Proteobacteria, PII is further subject to reversible uridylylation according to the intracellular levels of glutamine, which reflect the cellular nitrogen status. A number of PII proteins have been shown to bind ADP and ATP in a competitive manner, suggesting that PII might act as an energy sensor. Here, we analyse the influence of the ADP/ATP ratio, 2-OG levels and divalent metal ions on in vitro uridylylation of the Azospirillum brasilense PII proteins GlnB and GlnZ, and on interaction with their targets AmtB, DraG and DraT. The results support the notion that the cellular concentration of 2-OG is a key factor governing occupation of the GlnB and GlnZ nucleotide binding sites by ATP or ADP, with high 2-OG levels favouring the occupation of PII by ATP. Both PII uridylylation and interaction with target proteins responded to the ADP/ATP ratio within the expected physiological range, supporting the concept that PII proteins might act as cellular energy sensors.