No abstract

The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of Mycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export-the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in Mycobacterium smegmatis, we demonstrate that secA1 is an essential gene. In contrast, secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature of secA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a ⌬secA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

The SecA protein is present in all bacteria, and it is a central component of the general Sec-dependent protein export pathway. An unusual property of Mycobacterium tuberculosis is the presence of two SecA proteins: SecA1, the essential "housekeeping" SecA, and SecA2, the accessory secretion factor. Here, we report that a ⌬secA2 mutant of M. tuberculosis was defective for growth in the early stages of low-dose aerosol infection of C57BL/6 mice, a time during which the bacillus is primarily replicating in macrophages. Consistent with this in vivo phenotype, we found that the ⌬secA2 mutant was defective for growth in macrophages from C57BL/6 mice. The ⌬secA2 mutant was also attenuated for growth in macrophages from phox ؊/؊ mice and from NOS2 ؊/؊ mice. These mice are defective in the reactive oxygen intermediate ( Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most successful bacterial pathogens of all time. The global burden of tuberculosis is at its highest level ever and includes an increasing number of multiple-drug-resistant M. tuberculosis infections (74). Novel antituberculosis prevention and treatment measures are needed to control this health crisis, and a complete understanding of M. tuberculosis pathogenesis will help achieve this goal.M. tuberculosis is inhaled and taken up by unactivated host macrophages, where it is able to replicate and survive. Much remains to be learned about how M. tuberculosis avoids host defenses in these macrophages. The processes involved are likely complex and multifactorial. Among the properties implicated in M. tuberculosis survival in macrophages are the abilities of the bacillus to block acidification of the phagocytic vacuole, to block phagosome-lysosome fusion, and to resist reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) (34, 67).The early innate immune response to M. tuberculosis includes Toll-like receptor (TLR) stimulation of macrophages (35,55). TLR signaling leads to the secretion of RNI and proinflammatory cytokines that include tumor necrosis factor alpha (TNF-␣) and interleukin-6 (IL-6). Later in infection, activation of host macrophages by an adaptive TH1 immune response occurs, leading to the inhibition of intracellular growth and control of M. tuberculosis infection. Gamma interferon (IFN-␥) is an important mediator of this response; it upregulates antimycobacterial processes and antigen presentation by macrophages (22). One of the antimycobacterial effectors induced by IFN-␥ is RNI, which is produced by the inducible nitric oxide synthase (NOS2), although other IFN-␥-dependent effector mechanisms exist (13,39,40). TNF-␣ is another cytokine that plays an integral role in host control of M. tuberculosis infection (6,8,23). Among the many properties reported for TNF-␣ is that it can synergize with IFN-␥ to induce potent antimycobacterial activity of macrophages (13,21,22).It is increasingly apparent that M. tuberculosis can limit the host immune response and macrophage activation and that this inhi...

Previously, we obtained a Legionella pneumophila mutant, NU208, that is hypersensitive to iron chelators when grown on standard Legionella media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known L. pneumophila siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and Hartmannella and Acanthamoeba amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome c maturation gene, ccmC. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome c. Phenotypic analysis of two additional, independently derived ccmC mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation. trans-complementation analysis of NU208 confirmed that L. pneumophila ccmC is required for cytochrome c production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although ccm genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a ccm gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the ccm locus from strain 130b identified the genes ccmA-H. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between ccmB and ccmC. Southern hybridizations indicated that the open reading frame within this element (ISLp 1) was present in multiple copies in some strains of L. pneumophila but was absent from others. These findings represent the first evidence for a transposable element in Legionella and the first identification of an L. pneumophila strain-specific gene.

In bacteria, the majority of exported proteins are transported by the general Sec pathway from their site of synthesis in the cytoplasm across the cytoplasmic membrane. The essential SecA ATPase powers this Sec-mediated export. Mycobacteria possess two nonredundant SecA homologs: SecA1 and SecA2. In pathogenic Mycobacterium tuberculosis and the nonpathogenic model mycobacterium Mycobacterium smegmatis, SecA1 is essential for protein export and is the "housekeeping" SecA, whereas SecA2 is an accessory SecA that exports a specific subset of proteins. In M. tuberculosis the accessory SecA2 pathway plays a role in virulence. In this study, we uncovered basic properties of the mycobacterial SecA2 protein and its pathway for exporting select proteins. By constructing secA2 mutant alleles that encode proteins defective in ATP binding, we showed that ATP binding is required for SecA2 function. SecA2 mutant proteins unable to bind ATP were nonfunctional and dominant negative. By evaluating the subcellular distribution of each SecA, SecA1 was shown to be equally divided between cytosolic and cell envelope fractions, whereas SecA2 was predominantly localized to the cytosol. Finally, we showed that the canonical SecA1 has a role in the process of SecA2-dependent export. The accessory SecA2 export system is important to the physiology and virulence of mycobacteria. These studies help establish the mechanism of this new type of specialized protein export pathway.

Legionella pneumophila is an intracellular pathogen of protozoa and alveolar macrophages. This bacterium contains a gene (pilD) that is involved in both type IV pilus biogenesis and type II protein secretion. We previously demonstrated that the PilD prepilin peptidase is crucial for intracellular infection by L. pneumophila and that the secreted pilD-dependent proteins include a metalloprotease, an acid phosphatase, an esterase/ lipase, a phospholipase A, and a p-nitrophenyl phosphorylcholine hydrolase. Since mutants lacking type IV pili, the protease, or the phosphorylcholine hydrolase are not defective for intracellular infection, we sought to determine the significance of the secreted acid phosphatase activity. Three mutants defective in acid phosphatase activity were isolated from a population of mini-Tn10-mutagenized L. pneumophila. Supernatants as well as cell lysates from these mutants contained minimal acid phosphatase activity while possessing normal levels of other pilD-dependent exoproteins. Genetic studies indicated that the gene affected by the transposon insertions encoded a novel bacterial histidine acid phosphatase, which we designated Map for major acid phosphatase. Subsequent inhibitor studies indicated that Map, like its eukaryotic homologs, is a tartratesensitive acid phosphatase. The map mutants grew within macrophage-like U937 cells and Hartmannella amoebae to the same degree as did wild-type legionellae, indicating that this acid phosphatase is not essential for L. pneumophila intracellular infection. However, in the course of characterizing our new mutants, we gained evidence for a second pilD-dependent acid phosphatase activity that, unlike Map, is tartrate resistant.

Many studies of Mycobacterium tuberculosis infection and immunity have used mouse models. However, outcomes of vaccination and challenge with M. tuberculosis in inbred mouse strains do not reflect the full range of outcomes seen in people. Previous studies indicated that the novel Diversity Outbred (DO) mouse population exhibited a spectrum of outcomes after primary aerosol infection with M. tuberculosis. Here, we demonstrate the value of this novel mouse population for studies of vaccination against M. tuberculosis aerosol challenge. Using the only currently licensed tuberculosis vaccine, we found that the DO population readily controlled systemic Mycobacterium bovis BCG bacterial burdens and that BCG vaccination significantly improved survival across the DO population upon challenge with M. tuberculosis. Many individual DO mice that were vaccinated with BCG and then challenged with M. tuberculosis exhibited low bacterial burdens, low or even no systemic dissemination, little weight loss, and only minor lung pathology. In contrast, some BCG-vaccinated DO mice progressed quickly to fulminant disease upon M. tuberculosis challenge. Across the population, most of these disease parameters were at most modestly correlated with each other and were often discordant. This result suggests the need for a multiparameter metric to better characterize “disease” and “protection,” with closer similarity to the complex case definitions used in people. Taken together, these results demonstrate that DO mice provide a novel small-animal model of vaccination against tuberculosis that better reflects the wide spectrum of outcomes seen in people. IMPORTANCE We vaccinated the Diversity Outbred (DO) population of mice with BCG, the only vaccine currently used to protect against tuberculosis, and then challenged them with M. tuberculosis by aerosol. We found that the BCG-vaccinated DO mouse population exhibited a wide range of outcomes, in which outcomes in individual mice ranged from minimal respiratory or systemic disease to fulminant disease and death. The breadth of these outcomes appears similar to the range seen in people, indicating that DO mice may serve as an improved small-animal model to study tuberculosis infection and immunity. Moreover, sophisticated tools are available for the use of these mice to map genes contributing to control of vaccination. Thus, the present studies provided an important new tool in the fight against tuberculosis.