Biochemical and immunological characterization of a cpn60.1 knockout mutant of Mycobacterium bovis BCG.

Pathogenic mycobacteria possess two homologous chaperones encoded by cpn60.1 and cpn60.2. Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study, we show that inactivation of the Mycobacterium bovis BCG cpn60.1 (Mb3451c) gene does not significantly affect bacterial growth in 7H9 broth, but that this knockout mutant (Δcpn60.1) forms smaller colonies on solid 7H11 medium than the parental and complemented strains. When growing on Sauton medium, the Δcpn60.1 mutant exhibits a thinner surface pellicle and is associated with higher culture filtrate protein content and, coincidentally, with less protein in its outermost cell envelope in comparison with the parental and complemented strains. Interestingly, in this culture condition, the Δcpn60.1 mutant is devoid of phthiocerol dimycocerosates, and its mycolates are two carbon atoms longer than those of the wild-type, a phenotype that is fully reversed by complementation. In addition, Δcpn60.1 bacteria are more sensitive to stress induced by H(2)O(2) but not by SDS, high temperature or acidic pH. Taken together, these data indicate that the cell wall of the Δcpn60.1 mutant is impaired. Analysis by 2D gel electrophoresis and MS reveals the upregulation of a few proteins such as FadA2 and isocitrate lyase in the cell extract of the mutant, whereas more profound differences are found in the composition of the mycobacterial culture filtrate, e.g. the well-known Hsp65 chaperonin Cpn60.2 is particularly abundant and increases about 200-fold in the filtrate of the Δcpn60.1 mutant. In mice, the Δcpn60.1 mutant is less persistent in lungs and, to a lesser extent, in spleen, but it induces a comparable mycobacteria-specific gamma interferon production and protection against Mycobacterium tuberculosis H37Rv challenge as do the parental and complemented BCG strains. Thus, by inactivating the cpn60.1 gene in M. bovis BCG we show that Cpn60.1 is necessary for the integrity of the bacterial cell wall, is involved in resistance to H(2)O(2)-induced stress but is not essential for its vaccine potential.

Effect of PstS sub-units or PknD deficiency on the survival of Mycobacterium tuberculosis.

The membrane-associated phosphate-specific transporter (Pst) complex is composed of four different proteins: PstS, PstC, PstA and PstB. The PstS component detects and binds Pi with high affinity; the PstA and PstC form transmembrane pores for Pi entry, while PstB provides energy through ATP hydrolysis. In the Mycobacterium tuberculosis genome, four different gene clusters encode three PstS, and two of each of the other sub-units. We used RT-PCR to show that these clusters represent at least three distinct operons. The pstS3-containing operon was the only one induced by lack of environmental Pi. To study the physiologic role of the different PstS sub-units and that of another potential Pi receptor, PknD, we constructed and complemented their knockout (KO) mutants. In Sauton medium, the PstS1-3 KO grew faster than the Wt or the PknD KO. Following 24 h of complete starvation, the PstS3 or PknD deficient strains died if exposed to Pi poor conditions while the PstS1 and PstS2 KO survived and still grew faster than the Wt strain. These results suggest that PstS1-3 may play a role in the regulation of M. tuberculosis growth or metabolism while PstS3 and PknD contribute to the survival of the bacteria in phosphate poor conditions.

IS1096-mediated DNA rearrangements play a key role in genome evolution of Mycobacterium smegmatis.

The acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content. In this study, comparative genomics has allowed us to infer the roles of genomic rearrangement and changes in both distribution and copy number of the insertion element, IS1096, in the evolution of Mycobacterium smegmatis mc2155 from its progenitor, M. smegmatis ATCC 607. Comparative analysis revealed that the ATCC 607 genome contains only 11 IS1096 elements against the 24 reported in mc2155. As mc2155 evolved, there was a considerable expansion in the copy number of IS1096 (+13) as well as duplication of a 56-kb fragment flanked on both sides by IS1096; concurrently, a single IS1096 element and its flank were deleted. This study demonstrates that insertion sequence (IS) expansion and IS-induced rearrangements such as duplication, deletion and shuffling are major forces driving genomic diversity and evolution.

Cloning, sequencing, and cell surface expression pattern of bovine immunoreceptor NKG2D and adaptor molecules DAP10 and DAP12.

NKG2D is an activating lectin-like receptor that initiates natural killer (NK) cell responses against transformed tumor cells expressing its ligands, i.e., molecules related to major histocompatibility complex (MHC) class I molecules. NKG2D lacks signaling elements in its cytoplasmic domain and can deliver stimulatory signals only in association with transmembrane adaptor proteins DAP10 or DAP12. The complementary DNAs (cDNAs) encoding the bovine homologues of NKG2D and the adaptor proteins DAP10 and DAP12 were cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from resting bovine peripheral blood mononuclear cells (PBMC) and sequenced. Comparison with human, pig, and mouse sequences showed that bovine NKG2D is most similar to pig NKG2D and short mouse NKG2D (NKG2D-S). Similar to its human, mouse, and pig homologues, the cDNA for bovine DAP10 codes for a phosphatidyl-inositol-3 (PI-3) kinase-binding site (YxxM) in its cytoplasmic region. Finally, similar to its human, mouse, and pig homologues, the cDNA encoding bovine DAP12 demonstrates one tyrosine-based activated motif (ITAM) in its cytoplasmic domain. Bovine NKG2D cell surface expression was analyzed by flow cytometry on HEK 293 cells transiently transfected with cDNA expression vectors encoding COOH-terminal polyhistidine-tagged NKG2D and NH(2)-terminal Flag-tagged DAP10 and DAP12. Confirming previous findings for short mouse NKG2D-S, bovine NKG2D immunoreceptor could associate with either DAP10 or DAP12 adaptor protein for its cell surface expression.

A derivative of Mycobacterium smegmatis mc(2)155 that lacks the duplicated chromosomal region.

The genome of Mycobacterium smegmatis mc(2)155 contains a 56kb duplicated region. We isolated a mutant of mc(2)155 lacking this duplication (DeltaDRKIN). This mutation did not affect the growth rate, surface properties or transformation efficiency of the organism, confirming the potential utility of DeltaDRKIN for the study of genes contained within the duplicated region.

Expression of a bioactive recombinant human interleukin-11 in chicken HD11 cell line.

To direct the synthesis and secretion of recombinant human interleukin-11 (rhIL-11) in chicken HD11 cells, a plasmid targeting the c-lysozyme gene has been constructed which contains the mature cytokine cDNA in frame with the lysozyme leader sequence. The upregulation of rhIL-11 mediated by LPS proves the knock-in of hIL-11 cDNA in the lysozyme gene. The bioactivity of the expressed protein is demonstrated and quantified with the hIL-11 dependent 7TD1 and B9 cell lines. The electrophoretic mobility, receptor binding properties and growth promoting effect of the chicken-derived cytokine are identical to those of a rhIL-11 expressed in Escherichia coli. These results describe the secretion of a biologically active rhIL-11 expressed by an avian cellular machinery.

Mycobacterium tuberculosis with disruption in genes encoding the phosphate binding proteins PstS1 and PstS2 is deficient in phosphate uptake and demonstrates reduced in vivo virulence.

By measuring phosphate uptake by Mycobacterium tuberculosis strains with the pstS1 and pstS2 genes genetically inactivated, we showed that these pstS genes encode high-affinity phosphate binding proteins. In a mouse infection model, both mutants were attenuated in virulence, suggesting that M. tuberculosis encounters limiting phosphate concentrations during its intracellular life span.

Characterization of a potent human interleukin-11 agonist.

Human interleukin-11 (hIL-11) is a multi-potential cytokine that is involved in numerous biological activities, such as haematopoiesis, osteoclastogenesis, neurogenesis and female fertility, and also displays anti-inflammatory properties. IL-11 is used clinically to treat chemotherapy-induced thrombocytopenia. Because of its broad spectrum of action, improved IL-11 agonists, as well as IL-11 antagonists, could be of interest for numerous clinical applications. IL-11 signalling is dependent on the formation of a tripartite ligand-receptor complex consisting of IL-11, the IL-11R (IL-11 receptor) alpha subunit (responsible for the specificity of the interaction) and gp130 (glycoprotein 130) receptor beta subunit (responsible for signal transduction). The interaction between IL-11 and IL-11Ralpha subunit occurs at its recently assigned site I. We have designed an IL-11 mutein whose hydrophobicity at site I has been increased. The mutein has been characterized in terms of structure, affinity, specificity and bioactivity. Electrophoretic analysis, gel filtration, IR spectroscopy and CD indicate that this new protein is more compact than wild-type IL-11. It binds to IL-11Ralpha with a three-fold-enhanced affinity, and retains the ability to recruit gp130 through site II. However, analysis of its biological activity revealed a complex pattern: although this mutein is 60-400-fold more active than wild-type IL-11 on the proliferation of 7TD1 murine hybridoma cell, it is less active than IL-11 on the proliferation of B9 cells, another murine hybridoma cell line. The results are interpreted on the basis of an IL-11 conformational change induced by the mutations, and the preferential use by the mutein of another unknown transducing receptor chain.

Specific identification of Listeria welshimeri and Listeria monocytogenes by PCR assays targeting a gene encoding a fibronectin-binding protein.

We have cloned and sequenced a Listeria welshimeri DNA fragment homologous to the previously described fibronectin-binding protein-encoding gene (fbp) of Listeria monocytogenes (P. Gilot, Y. Jossin, and J. Content, J. Med. Microbiol., 49:887-896, 2000). This L. welshimeri DNA fragment expresses a 24.8-kDa protein that binds to human fibronectin. Based on the fbp sequences, we developed novel PCR assays for the identification of L. welshimeri and L. monocytogenes.

Engineering and use of (32)P-labeled human recombinant interleukin-11 for receptor binding studies.

Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human IL-11 (FP Delta IL-11). In this molecule, a specific phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with (32)P. A high radiological specific activity (250,000 c.p.m x ng(-1) of protein) was obtained with the retention of full biological activity of the protein. The binding of (32)P-labeled FP Delta IL-11 to Ba/F3 cells stably transfected with plasmids encoding human IL-11 receptors alpha and beta chains (IL-11R alpha and gp130) was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure, expression and regulation.

Cloning, sequencing and characterisation of a Listeria monocytogenes gene encoding a fibronectin-binding protein.

Listeria monocytogenes is a gram-positive, non-sporulating food-borne pathogen of man and animals that is able to invade many eukaryotic cells. L. monocytogenes possesses several proteins that bind fibronectin. In this study, an L. monocytogenes DNA library in pUC19 was screened with fibronectin and a gene encoding a 24.6-kDa fibronectin-binding protein (Fbp) was isolated and sequenced. Transcripts of the fbp gene were found in wild-type, in deltaprfA, and PrfA-S183A strains, despite the presence of a 'PrfA-like' box around its ribosome-binding site. The fbp gene was found to be present in all tested isolates of the species L. monocytogenes and a homologous DNA fragment was amplified in L. welshimeri. No homologies between the fbp gene and its translation product with any other DNA or proteins deposited in databanks were found. Restriction endonuclease-PCR (RE-PCR) showed that the fbp gene displays a degree of allelic variation among isolates of L. monocytogenes, whereas the corresponding amplified fragment of L. welshimeri seems to be monomorphic among isolates of this species. RE-PCR with Hha I, Dde I or Taq I produced DNA banding profiles specific for each of these two species, allowing their identification.