Assessment of the serodiagnostic potential of nine novel proteins from Mycobacterium tuberculosis.

To identify antigens that would improve the accuracy of serological diagnosis of active tuberculosis, we cloned the genes encoding nine potentially immunogenic secreted or surface-associated proteins of Mycobacterium tuberculosis. Recombinant proteins were reacted with sera from HIV-negative individuals with extrapulmonary tuberculosis (EP-TB) or HIV-positive individuals with pulmonary tuberculosis (TBH). Specific and high level antibody responses were obtained for four recombinant proteins, of which antigen GST-822 was recognized by 60% of EP-TB and 42% of TBH and antigen MBP-506 was recognized by 45% of EP-TB and 61% of TBH. These results suggest that these proteins are strong candidates as subunits in a polyvalent serodiagnostic test.

Heat-labile proteases in molecular biology applications.

Thermolabile proteases were identified in three Gram-negative psychrotrophic bacteria. The protease from the psychrotrophic strain A9 was purified and its application to common molecular biology techniques was demonstrated. Heat-stable molecular biology enzymes (Taq polymerase and PvuII) were digested by a heat-labile protease, which was then inactivated by a mild heat treatment. The clear benefit of using heat-labile proteases arises in situations where further reactions may be accomplished without an intermediate purification step, thereby saving time and avoiding the possibility of product loss.

Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis.

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.

Identification of novel immunogenic Mycobacterium tuberculosis peptides that stimulate mononuclear cells from immune donors.

Proteins which are secreted or associated with the cell envelope of Mycobacterium tuberculosis may contain protective T-cell epitopes. Prior to this study, a recombinant clone bank of enzymatically active M. tuberculosis-alkaline phosphatase fusions, were screened for immunogenicity in a murine T-cell model. Five of these were selected for further study, and the IFN-gamma secretion and proliferation of human PBMC from purified protein derivative- (PPD)-positive and PPD-negative donors were measured in response to oligopeptides, Mtb-PhoA fusions and one full-length protein. Epitopes from four of the five selected antigens were immunoreactive in the human model and corresponded to cytochrome d ubiquinol oxidase, cytochrome c oxidase subunit II, MTV005.02 and MTV033.08. Thus, this strategy identified novel human immunogenic peptides as possible candidates for a subunit vaccine.

The respiratory burst-inhibiting acid phosphatase AcpA is not essential for the intramacrophage growth or virulence of Francisella novicida.

Acid phosphatases capable of inhibiting the respiratory burst of neutrophils have been identified in certain intracellular pathogens. Here we evaluate the role of AcpA, a respiratory burst-inhibiting acid phosphatase of Francisella, in the virulence and intracellular growth of this organism. An F. novicida acpA null mutant was created and found to exhibit wild-type growth kinetics in both cell-line and inflammatory mouse macrophages. The acpA mutant also shows wild-type replication in the spleens of experimentally infected mice. These data suggest that AcpA is not essential for the intracellular growth or virulence of F. novicida.

An erythromycin resistance cassette and mini-transposon for constructing transcriptional fusions to cat.

A new cassette (Er-Cm cassette) and mini-transposon (mTn) (TnMaxErCm) based on the previously described mTn, TnMax2 [Haas et al., Gene 130, 23-31.], have been constructed. The cassette and mTn make use of an erythromycin resistance (ErR) marker encoded by ermC'. Both the Er-Cm cassette and TnMaxErCm also carry a promoterless cat gene to allow the construction of transcriptional fusions and the measurement of transcriptional activity by assaying for chloramphenicol acetyltransferase. We show the function of these genetic elements by analyzing the regulation of expression of the mglA gene of Francisella novicida and by using TnMaxErCm to probe for promoter activity within an F. novicida recombinant clone. The reporter cassette and mTn described here further expand the family of TnMax transposons and facilitate the study of gene expression in organisms where direct Tn mutagenesis methods are unavailable.

MglA and MglB are required for the intramacrophage growth of Francisella novicida.

Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated that are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for both the intracellular growth defect and the colony morphology on LB (X-p) media. The locus consists of an apparent operon of two genes, designated mgIAB, for Macrophage Growth Locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 or LB (X-p) media. Sequencing on mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Cell fractionation studies revealed several differences in the protein profiles of mgI mutants compared with wild-type F. novicida. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may affect the expression of genes whose products contribute to survival and growth within macrophages.

Temperature-sensitive lesions in the Francisella novicida valA gene cloned into an Escherichia coli msbA lpxK mutant affecting deoxycholate resistance and lipopolysaccharide assembly at the restrictive temperature.

The valAB locus of Francisella novicida has previously been found to be highly similar at the deduced amino acid level to msbA lpxK of Escherichia coli. Both ValA and MsbA are members of the superfamily of ABC transporters, and they appear to have similar functions. In this study we describe the isolation of a temperature-sensitive valAB locus. DNA sequence analysis indicates that the only changes to the ValAB deduced amino acid sequence are changes of S453 to an F and T458 to an I in ValA. E. coli strains defective in msbA and expressing temperature-sensitive ValA rapidly ceased growth when shifted from a permissive temperature to a restrictive temperature. After 1 h at the restrictive temperature, cells were much more sensitive to deoxycholate treatment. To test the hypothesis that ValA is responsible for the transport or assembly of lipopolysaccharide, we introduced gseA, a Kdo (3-deoxy-D-manno-octulosonic acid) transferase from Chlamydia trachomatis, into a strain with a temperature-sensitive valA allele and a nonfunctional msbA locus. These recombinants were defective in cell surface expression of the chlamydial genus-specific epitope within 15 min of a shift to the nonpermissive temperature. Also, there was enhanced association of the epitope with the inner membrane after a shift to the nonpermissive temperature. Thus, we propose that ValA is involved in the transport of lipopolysaccharide to the outer membrane.

Suppression of Francisella tularensis growth in the rat by co-infection with F. novicida.

We have previously shown that when cultured in vitro, peritoneal rat macrophages infected with Francisella novicida spontaneously release nitric oxide in sufficient quantities to inhibit bacterial growth. However, it is not known whether F. novicida can have a similar antimicrobial effect in vivo. Here we show that a co-infection of F. novicida with Francisella tularensis can suppress the number of F. tularensis cells in rat spleens by as much as 100-fold.

Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family.

The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species.

Phase variation in Francisella tularensis affecting intracellular growth, lipopolysaccharide antigenicity and nitric oxide production.

Many microbial pathogens, such as Mycobacterium spp. and Salmonella spp., use macrophage intracellular growth or antigenic variation as mechanisms for avoiding the host immune system. In this work we present evidence to show that the intracellular pathogen Francisella tularensis uses phase variation to alter antigenicity and the host macrophage nitric oxide response simultaneously, thereby modulating its intracellular growth. The lipopolysaccharide (LPS) and lipid A of F. tularensis fails to stimulate production of significant levels of nitric oxide (NO) by rat macrophages. However, spontaneous variants of F. tularensis expressing an antigenically distinct LPS induce rat macrophages to produce increased levels of NO, thereby suppressing microbial intramacrophage growth. Similarly, lipid A isolated from these variants stimulates increased levels of NO production. A reverse phase shift can occur, which returns the LPS to the original antigenic form, reduces NO production, and restores intramacrophage growth. These findings represent the first demonstration of a phase-variation phenomenon which modulates intracellular growth and an innate immune response. Furthermore, these results suggest that a microbial pathogen can exploit macrophage NO production for its own benefit, perhaps by prolonging the host-pathogen association during the acute phase of disease or during the process of establishing a carrier state.

Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis.

Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12-myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase.

New vectors for the in vitro generation of alkaline phosphatase fusions to proteins encoded by G+C-rich DNA.

Phagemid vectors were constructed to allow fusions of alkaline phosphatase to proteins encoded by G+C-rich DNA, by engineering a BstBI site (TT/CGAA) in front of a phoA gene that lacks an encoded signal peptide. Three vectors (pJDT1, pJDT2 and pJDT3), each with phoA in a different reading frame with respect to the BstBI site, were produced; a lacP region is present in each plasmid upstream of the BstBI site. The presence of the BstBI site allows the random cloning of G+C-rich DNA digested with a number of restriction enzymes that generate cohesive ends.

Isolation of a Francisella tularensis mutant that is sensitive to serum and oxidative killing and is avirulent in mice: correlation with the loss of MinD homologue expression.

We constructed mutant strains of Francisella tularensis biotype novicida by insertional mutagenesis with a kanamycin resistance (KmR) cassette. One mutant, KEM7, was defective for survival in macrophages in comparison with the wild-type (WT) strain and a random insertion strain, KEM21. While all three strains exhibited intracellular growth, the number of viable KEM7 present after 24-48 h of infection was approximately 10 times less than that of WT or KEM21. This observation was apparently due to a reduced number of viable KEM7 associated with the macrophages one hour after phagocytosis. KEM7 was approximately 3 times more susceptible than WT or KEM21 to killing by the products of the xanthine-xanthine oxidase reaction or by hydrogen peroxide. KEM7 was also found to be susceptible to killing by serum, whereas WT and KEM21 were resistant. Upon intravenous inoculation of C57BL/6 mice, the number of KEM7 in the livers and spleens 48 h post-infection was found to be 1,000- to 10,000-times less than that of either KEM21 or WT. DNA sequence analysis at the KmR insertion site suggested that the F. tularensis homologue of minD had been interrupted. Western immunoblot analysis confirmed the presence of a MinD homologue in F. tularensis WT and KEM21, and demonstrated its absence in KEM7.

Serum-sensitive mutation of Francisella novicida: association with an ABC transporter gene.

Francisella novicida is a facultative intracellular pathogen that can survive and grow in macrophages by preventing phagolysosomal fusion. In this study in vitro cassette mutagenesis was used to generate a library of insertion mutants of F.novicida. Two related mutants, KM14 and KM14S, initially identified as defective for growth in macrophages, were found to be sensitive to serum. These mutants were also found to grow approximately 1000-fold less well in the livers and spleens of infected mice. We cloned a genetic locus that was presumably mutagenized in these mutants and found that it included genes that had high similarity in their deduced amino acid sequence to those of msbA and orfE of Escherichia coli. The former is a member of the superfamily of ABC transporter proteins. We named the corresponding genes in F. novicida, valAB. Integration of a cloned valAB locus into the chromosome of KM14S partially restored the serum resistance phenotype found in wild-type F. novicida.

Lipoarabinomannan from Mycobacterium tuberculosis modulates the generation of reactive nitrogen intermediates by gamma interferon-activated macrophages.

Lipoarabinomannan derived from the virulent Erdman strain and a rapidly growing, laboratory-attenuated strain of Mycobacterium tuberculosis were evaluated for their ability to modulate the production of nitric oxide (NO) by macrophages activated with IFN-gamma or IFN-gamma and LPS. It was observed that in macrophages pretreated with 100 micrograms ml-1 LAM, the NO induced by IFN-gamma alone was augmented while the NO induced IFN-gamma and LPS was reduced. LAM was also shown to synergize with IFN-gamma in the induction of NO, with AraLAM from the attenuated strain exhibiting greater potency than ManLAM from the Erdman strain. Despite the modulation of NO production, LAM did not affect the IFN-gamma-induced macrophage growth inhibition of Francisella tularensis LVS, an organism whose growth inhibition in activated macrophages is dependent upon NO.

Arg276 of GseA, a Chlamydia trachomatis Kdo transferase, is required for the synthesis of the chlamydial genus-specific epitope in Escherichia coli.

GseA is an enzyme from Chlamydia trachomatis that can catalyse the addition of three 3-deoxy-D-manno-octulosonic acid (Kdo) residues onto lipid A precursors. GseA is similar, and in a few stretches identical, in its amino acid sequence to KdtA, an Escherichia coli Kdo transferase. In this study we altered an amino acid of GseA in a region that is identical between GseA and KdtA to test its importance in the structure or catalytic activity of GseA. We found that when Arg276 was changed to Lys, Ile or Ser, GseA activity was lost, suggesting an enzymatic role for this amino acid residue.

A novel 3-deoxy-D-manno-octulosonic acid transferase from Chlamydia trachomatis required for expression of the genus-specific epitope.

DNA cloned from Chlamydia trachomatis is able to direct the formation of the genus-specific lipopolysaccharide epitope of chlamydiae in enteric Gram-negative bacteria. We now demonstrate that a single C. trachomatis gene (gseA) is sufficient to impart this trait to Escherichia coli. The deduced amino acid sequence of gseA shows 23% identity (66% similarity) to kdtA, an E. coli gene that codes for a bifunctional enzyme catalyzing the addition of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A precursors (Clementz, T., and Raetz, C. R. H. (1991) J. Biol. Chem. 266, 9687-9696). Extracts of E. coli expressing gseA transfer at least one additional Kdo unit from CMP-Kdo to precursors already bearing the two Kdo residues attached by the kdtA gene product. Introduction of gseA into an E. coli mutant with a thermolabile kdtA gene product endows cell extracts with the ability to transfer not only the third but also the first two Kdos to lipid A precursors, demonstrating that the C. trachomatis enzyme is at least trifunctional. Given the similarities of these two Kdo transferases and the essentiality of Kdo in Gram-negative bacteria, lipopolysaccharide biosynthesis may be a target for development of novel drugs effective against chlamydiae.

Growth inhibition of Francisella tularensis live vaccine strain by IFN-gamma-activated macrophages is mediated by reactive nitrogen intermediates derived from L-arginine metabolism.

We have examined the abilities of the recombinant murine lymphokines IFN-gamma, granulocyte-macrophage (GM)-CSF, and IL-4 to stimulate the in vitro antimicrobial activity of macrophages against the live vaccine strain (LVS) of Francisella tularensis. Resident peritoneal macrophages from C57BL/6 strain mice were cultured overnight with IFN-gamma, GM-CSF, or IL-4, and then infected with LVS. In macrophages treated with IFN-gamma, the growth of LVS was suppressed by a factor of 100- to 1000-fold in comparison with untreated cells. This effect was dose-dependent and was enhanced by the addition of LPS. In contrast, macrophages treated with either GM-CSF or IL-4 exhibited no such enhanced antitularemic activity, even in the presence of LPS. Because reactive nitrogen intermediates derived from L-arginine metabolism have been implicated in the killing of various infectious organisms, we evaluated the possibility that such a mechanism might contribute to the antitularemic activity of IFN-gamma-stimulated macrophages. Macrophages were treated with NG-monomethyl-L-arginine (NMMA), an inhibitor of L-arginine metabolism in mammalian cells, during the activation procedure and throughout the course of infection. NMMA had no effect on the growth of LVS in unstimulated macrophages. In macrophages activated with IFN-gamma, however, NMMA suppressed their capacity to inhibit LVS growth. This effect was proportional to the dose of NMMA added and reversible by supplementing the medium with additional L-arginine, and there was a direct correlation between the production of nitrite by activated macrophages and their ability to inhibit LVS growth. Furthermore, the growth of LVS was inhibited by nitrogen metabolites in a cellfree system. The results of this study indicate that the mechanism of action of IFN-gamma on the resistance of macrophages to LVS growth is related, at least in part, to the production of reactive nitrogen metabolites.