Listeria monocytogenes-infected phagocytes can initiate central nervous system infection in mice.

Listeria monocytogenes-infected phagocytes are present in the bloodstream of experimentally infected mice, but whether they play a role in central nervous system (CNS) invasion is unclear. To test whether bacteria within infected leukocytes could establish CNS infection, experimentally infected mice were treated with gentamicin delivered by surgically implanted osmotic pumps. Bacterial inhibitory titers in serum and plasma ranged from 1:16 to 1:256, and essentially all viable bacteria in the bloodstream of treated mice were leukocyte associated. Nevertheless, CNS infection developed in gentamicin-treated animals infected intraperitoneally or by gastric lavage, suggesting that intracellular bacteria could be responsible for neuroinvasion. This was supported by data showing that 43.5% of bacteria found with blood leukocytes were intracellular and some colocalized with F-actin, indicating productive intracellular parasitism. Experiments using an L. monocytogenes strain containing a chromosomal actA-gfpuv-plcB transcriptional fusion showed that blood leukocytes were associated with intracellular and extracellularly bound green fluorescent protein-expressing (GFP+) bacteria. Treatment with gentamicin decreased the numbers of extracellularly bound GFP+ bacteria significantly but did not affect the numbers of intracellular GFP+ bacteria, suggesting that the latter were the result of intercellular spread of GFP+ bacteria to leukocytes. These data demonstrate that infected leukocytes and the intracellular L. monocytogenes harbored within them play key roles in neuroinvasion. Moreover, they suggest that phagocytes recruited to infected organs such as the liver or spleen are themselves parasitized by intercellular spread of L. monocytogenes and then reenter the bloodstream and contribute to the systemic dissemination of bacteria.

Sequence variations within PrfA DNA binding sites and effects on Listeria monocytogenes virulence gene expression.

Reporter gene fusions were used to investigate the contributions of PrfA DNA binding sites to Listeria monocytogenes virulence gene expression. Our results suggest that the DNA sequence of PrfA binding sites determines the levels of expression of certain virulence genes, such as hly and mpl. Other virulence genes, such as actA and plcB, may depend upon additional factors for full regulation of gene expression.

Mycobacterial infection of macrophages results in membrane-permeable phagosomes.

Cell-mediated immunity is critical for host resistance to tuberculosis. T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacterial infection. Mice genetically deficient in the generation of MHC class I and class Ia responses are susceptible to mycobacterial infection. Although soluble protein antigens are generally presented by macrophages to T cells through MHC class II molecules, macrophages infected with Mycobacterium tuberculosis or bacille Calmette-Guerin have been shown to facilitate presentation of ovalbumin through the MHC class I presentation pathway via a TAP-dependent mechanism. How mycobacteria, thought to reside within membrane-bound vacuoles, facilitate communication with the cytoplasm and enable MHC class I presentation presents a paradox. By using confocal microscopy to study the localization of fluorescent-tagged dextrans of varying size microinjected intracytoplasmically into macrophages infected with bacille Calmette-Guerin expressing the green fluorescent protein, molecules as large as 70 kilodaltons were shown to gain access to the mycobacterial phagosome. Possible biological consequences of the permeabilization of vacuolar membranes by mycobacteria would be pathogen access to host cell nutrients within the cytoplasm, perhaps contributing to bacterial pathogenesis, and access of microbial antigens to the MHC class I presentation pathway, contributing to host protective immune responses.

Examination of Listeria monocytogenes intracellular gene expression by using the green fluorescent protein of Aequorea victoria.

The ActA protein of Listeria monocytogenes is an essential virulence factor and is required for intracellular bacterial motility and cell-to-cell spread. plcB, cotranscribed with actA, encodes a broad-specificity phospholipase C that contributes to lysis of host cell vacuoles and cell-to-cell spread. Construction of a transcriptional fusion between actA-plcB and the green fluorescent protein gene of Aequorea victoria has facilitated the detailed examination of patterns of actA/plcB expression within infected tissue culture cells. actA/plcB expression began approximately 30 min postinfection and was dependent upon entry of L. monocytogenes into the host cytosol. L. monocytogenes Deltahly mutants, which are unable to escape from host cell vacuoles, did not express actA/plcB at detectable levels within infected tissue culture cells; however, complementation of the hly defect allowed entry of the bacteria into the host cytoplasm and subsequent actA/plcB expression. These results emphasize the ability of L. monocytogenes to sense the different host cell compartment environments encountered during the course of infection and to regulate virulence gene expression in response.

Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria.

Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. The assembly of these colonization factors is a complex process. This report describes a new pilus-assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre-pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus-assembly protein PilC of Pseudomonas aeruginosa and other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide of M(r) 38 kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group B polysaccharide-producing strain of N. meningitidis (Mc). The results indicate that the pilG genes and genetic organization at these loci in Gc and Mc are extremely conserved. Hybridization studies strongly suggest that pilG-related genes exist in commensal Neisseria species and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Mc chromosomes by allelic replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin-gene alterations or to polarity exerted on pilD expression. The results indicated that PilG exerts its own independent role in neisserial pilus biogenesis.

Characterization of the pilF-pilD pilus-assembly locus of Neisseria gonorrhoeae.

Expression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host. Several N. gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems. We report here the isolation and characterization of transposon insertion mutants in N. gonorrhoeae whose phenotypes indicate that the N. gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis. Mutants lacking the pilD gene product, a pre-pilin peptidase, were unable to process the pre-pilin subunit into pilin and thus were non-piliated. pilF mutants processed pilin but did not assemble the mature subunit. Both classes of mutants released S-pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly. In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype. Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression. orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide-binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans. Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili. Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.

Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo.

The PrfA transcriptional activator is an essential determinant of Listeria monocytogenes pathogenesis. prfA expression is governed by three differentially regulated promoters: prfAP1 and prfAP2, which are located immediately upstream of prfA coding sequences, and the adjacent plcA promoter via the generation of a plcA-prfA read-through transcript. A series of promoter deletion mutants were constructed to assess the roles of prfAP1 and prfAP2. Elimination of either prfAP1 or prfAP2 resulted in altered regulation of PrfA-regulated genes after in vitro growth. However, these mutants were fully virulent both in an animal model and in tissue culture models of infection, suggesting that the two prfA promoters are functionally redundant in vivo. In contrast, a mutant lacking both prfAP1 and prfAP2 was 100-fold less virulent and was delayed in escape from the host vacuole. Once in the host cytoplasm, however, the double mutant was apparently normal in cell-to-cell spread.

Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread.

The prfA gene product is a transcriptional activator of Listeria monocytogenes determinants of pathogenicity. In this study, we provide direct evidence that the PrfA protein is a site-specific DNA-binding protein. Additionally, we describe the characterization of two classes of L. monocytogenes mutants which contain transposon insertions either in the prfA structural gene (exemplified by strain DP-L1075) or within the prfA promoter region (exemplified by strain DP-L973). Both mutants are completely avirulent and secrete greatly reduced levels of listeriolysin O and phosphatidylinositol-specific phospholipase C, and both are fully complemented by the introduction of prfA on a multicopy plasmid. The behaviors of the two mutants differ markedly within cultured macrophages. Following infection, no cytoplasmic growth was observed for DP-L1075 whereas DP-L973 escaped from the phagosome and grew in the cell cytoplasm. However, DP-L973 was defective in nucleation of actin filaments and spread to adjacent cells. Transcription of prfA in DP-L973 was directed from a single, previously unidentified promoter (prfAp2) located close to the prfA initiation codon. This promoter is therefore capable of providing sufficient prfA expression for escape from the host cell vacuole but is insufficient for wild-type levels of bacterially induced actin polymerization and cell-to-cell spread. Transcription directed from both prfAp1 and prfAp2 promoters was increased in the absence of a functional prfA gene product, suggesting that PrfA protein contributes to down-regulating its own expression.

Transcriptional activation of the Listeria monocytogenes hemolysin gene in Bacillus subtilis.

The prfA gene of Listeria monocytogenes was recently reported to be required for expression of hly, which encodes a pore-forming hemolysin essential for pathogenicity (M. Leimeister-Wachter, C. Haffner, E. Domann, W. Goebel, and T. Chakraborty, Proc. Natl. Acad. Sci. USA 87:8336-8340, 1990). We demonstrate here that a hly-lacZ fusion introduced into Bacillus subtilis is strongly activated when the prfA gene product is supplied in trans under the control of an isopropyl-beta-D-thiogalactopyranoside-inducible promoter, Pspac. Moreover, the PrfA-dependent activation of hly is abolished by point mutations in a 14-bp DNA palindromic sequence present in the 5' upstream region of hly. This indicates that PrfA is both necessary and sufficient for hly transcriptional activation and establishes the palindrome as the likely target sequence for PrfA interaction. The presence of a palindrome in the upstream regions of three additional L. monocytogenes genes clustered near hly suggests that PrfA may serve as a transcriptional activator for a major virulence regulon of L. monocytogenes. In addition, the ability of PrfA to activate its target promoters effectively in B. subtilis suggests that further analysis of this regulon and perhaps other aspects of L. monocytogenes gene regulation might be carried out in part through reconstruction experiments in B. subtilis.

Site-directed mutagenesis of the RecA protein of Escherichia coli. Tyrosine 264 is required for efficient ATP hydrolysis and strand exchange but not for LexA repressor inactivation.

The role of Tyr264 in nucleotide binding and hydrolysis catalyzed by the RecA protein of Escherichia coli was investigated by constructing Gly, Ser, and Phe substitution mutations using oligonucleotide-directed mutagenesis. The corresponding mutant recA genes neither restored resistance to killing by ultraviolet irradiation nor increased homologous recombination in a recA strain. The purified RecA(Gly264) protein was unable to bind nucleotide, hydrolyze ATP, or form stable ternary complexes with adenosine 5'-O-thiotriphosphate and DNA although the mutant protein bound DNA normally in the absence of nucleotide. The RecA (Phe264) and RecA(Ser264) proteins hydrolyzed ATP poorly and the rates were reduced approximately 8- and 18-fold, respectively. Although capable of low levels of ATP hydrolysis, neither the RecA(Phe264) nor the RecA(Ser264) protein promoted DNA pairing or strand exchange reactions in vitro. Furthermore, these mutant RecA proteins were impaired in their ability to form salt-resistant ternary complexes with adenosine 5'-O-thiotriphosphate) and DNA as judged by filter binding. Nevertheless, nucleoprotein complexes formed with either RecA(Phe264) or RecA(Ser264) protein directed efficient cleavage of LexA repressor in vitro. These results demonstrate that Tyr264 is required for efficient ATP hydrolysis and for homologous pairing of DNA but does not participate in activating RecA protein for LexA repressor autodigestion.

LexA-independent expression of a mutant mucAB operon.

pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene products of which are required for the SOS-dependent processing of damaged DNA necessary for most mutagenesis. Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit, with LexA acting as a direct repressor. pGW16 is a pKM101 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increase in spontaneous mutation rate. In this report, we show that pGW16 differs from pKM101 in being able to enhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3 host. The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includes the mucAB coding region and approximately 0.6 kb of the 5'-flanking region. We determined the sequence of a 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in that region, a G.C-to-A.T transition that alters a sequence homologous to known LexA-binding sites. DNA gel shift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation, whereas a fragment containing the wild-type sequence is efficiently bound by LexA. This mutation also alters an overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closer to the consensus sequence. The observation that the synthesis of pGW16-encoded mucAB proteins in maxicells is increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmid suggests that this mutation also increases the activity of the mucAB promoter.

Protein carboxyl methyltransferase selectively modifies an atypical form of calmodulin. Evidence for methylation at deamidated asparagine residues.

Prolonged incubation of native bovine brain calmodulin with S-adenosyl-L-[methyl-3H]methionine and protein carboxyl methyltransferase results in maximal methylation of only 1-2% of the calmodulin molecules. In contrast, calmodulin which has been subjected to a prior alkaline treatment (0.1 M NH4OH, 37 degrees C for 3 h) can be methylated to a level of 30-50%. This treatment is known to be effective in deamidating certain asparagine residues which lie in unstable sequences, particularly -Asn-Gly- sequences. Bovine brain calmodulin has three such sequences (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). The enhancement of methylation by alkaline treatment is substantially reduced if calmodulin is first reacted with bis-(I,I-trifluoroacetoxy)iodobenzene, a reagent which converts the carboxamide group of asparagine and glutamine residues to the corresponding primary amines. Thus, protein carboxyl methyltransferase selectively modifies an abnormal form of calmodulin that is probably deamidated. These findings suggest that protein carboxyl methylation may play a role in the disposition of abnormal proteins which contain atypical, isomerized aspartyl residues arising via spontaneous deamidation of unstable asparagines.