Identification of Mycobacterium marinum macrophage infection mutants

Mycobacterium marinum is an important pathogen of humans, amphibians and fish. Most pathogenic mycobacteria, including M. marinum, infect, survive and replicate primarily intracellularly within macrophages. We constructed a transposon mutant library in M. marinum using Tn5367 delivered by phage transduction in the shuttle phasmid phAE94. We screened 529 clones from the transposon library directly in macrophage infection assays. All clones were screened for their ability to initially infect macrophages as well as survive and replicate intracellularly. We identified 19 mutants that fit within three classes: class I) defective for growth in association with macrophages (42%), class II) defective for macrophage infection (21%) and class III) defective for infection of and growth in association with macrophages (37%). Although 14 of the macrophage infection mutants (Mim) carry insertions in genes that have not been previously identified, five are associated with virulence of mycobacteria in animal models. These observations confirm the utility of mutant screens directly in association with macrophages to identify new virulence determinants in mycobacteria. We complemented four of the Mim mutants with their M. tuberculosis homologue, demonstrating that secondary mutations are not responsible for the observed defect in macrophage infection. The genes we identified provide insight into the molecular mechanisms of macrophage infection by M. marinum.     

Transposon mutagenesis of Mycobacterium marinum identifies a locus linking pigmentation and intracellular survival

Pathogenic mycobacteria survive and replicate within host macrophages, but the molecular mechanisms involved in this necessary step in the pathogenesis of infection are not completely understood. Mycobacterium marinum has recently been used as a model for aspects of the pathogenesis of tuberculosis because of its close genetic relationship to Mycobacterium tuberculosis and because of similarities in the pathology and course of infection caused by this organism in its natural hosts, fish and frogs, with tuberculosis in humans. In order to advance the utility of the M. marinum model, we have developed efficient transposon mutagenesis of the organism by using a Drosophila melanogaster mariner-based transposon. To determine the efficiency of transposition, we have analyzed pigmentation mutants from the transposon mutant library. In addition to insertions in four known genes in the pathway of pigment biosynthesis, two insertions in novel genes were identified in our mutant library. One of these is in a putative inhibitor of the carotenoid biosynthesis pathway. The second unexpected insertion is in an intergenic region between two genes homologous to Rv2603c and Rv2604c of M. tuberculosis. In addition to a pigmentation defect, this mutant showed increased susceptibility to singlet oxygen and grew poorly in murine macrophages. Complementation with M. tuberculosis genomic DNA encompassing Rv2603c to Rv2606c corrected the pigmentation and growth defects of the mutant. These data demonstrate the utility of mariner-based transposon mutagenesis of M. marinum and that M. marinum can be used to study the function of M. tuberculosis genes involved in intracellular survival and replication.     

The Mycobacterium tuberculosis SecA2 system subverts phagosome maturation to promote growth in macrophages

The ability of Mycobacterium tuberculosis to grow in macrophages is critical to the virulence of this important pathogen. One way M. tuberculosis is thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest by M. tuberculosis is not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest for M. tuberculosis growth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. In M. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2 mutant of M. tuberculosis were more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-type M. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2 mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2 mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest for M. tuberculosis growth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.     

The PmrA/PmrB two-component system of Legionella pneumophila is a global regulator required for intracellular replication within macrophages and protozoa

To examine the role of the PmrA/PmrB two-component system (TCS) of Legionella pneumophila in global gene regulation and in intracellular infection, we constructed pmrA and pmrB isogenic mutants by allelic exchange. Genome-wide microarray gene expression analyses of the pmrA and pmrB mutants at both the exponential and the postexponential phases have shown that the PmrA/PmrB TCS has a global effect on the expression of 279 genes classified into nine groups of genes encoding eukaryotic-like proteins, Dot/Icm apparatus and secreted effectors, type II-secreted proteins, regulators of the postexponential phase, stress response genes, flagellar biosynthesis genes, metabolic genes, and genes of unknown function. Forty-one genes were differentially regulated in the pmrA or pmrB mutant, suggesting a possible cross talk with other TCSs. The pmrB mutant is more sensitive to low pH than the pmrA mutant and the wild-type strain, suggesting that acidity may trigger this TCS. The pmrB mutant exhibits a significant defect in intracellular proliferation within human macrophages, Acanthamoeba polyphaga, and the ciliate Tetrahymena pyriformis. In contrast, the pmrA mutant is defective only in the ciliate. Despite the intracellular growth defect within human macrophages, phagosomes harboring the pmrB mutant exclude late endosomal and lysosomal markers and are remodeled by the rough endoplasmic reticulum. Similar to the dot/icm mutants, the intracellular growth defect of the pmrB mutant is totally rescued in cis within communal phagosomes harboring the wild-type strain. We conclude that the PmrA/PmrB TCS has a global effect on gene expression and is required for the intracellular proliferation of L. pneumophila within human macrophages and protozoa. Differences in gene regulation and intracellular growth phenotypes between the pmrA and pmrB mutant suggests a cross talk with other TCSs.     

The SecA2 secretion factor of Mycobacterium tuberculosis promotes growth in macrophages and inhibits the host immune response

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 DeltasecA2 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 DeltasecA2 mutant was defective for growth in macrophages from C57BL/6 mice. The DeltasecA2 mutant was also attenuated for growth in macrophages from phox(-/-) mice and from NOS2(-/-) mice. These mice are defective in the reactive oxygen intermediate (ROI)-generating phagocyte oxidase and the reactive nitrogen intermediate (RNI)-generating inducible nitric oxide synthase, respectively. This indicated a role for SecA2 in the intracellular growth of M. tuberculosis that is independent of protecting against these ROIs or RNIs. Macrophages infected with the DeltasecA2 mutant produced higher levels of tumor necrosis factor alpha, interleukin-6, RNI, and gamma interferon-induced major histocompatibility complex class II. This demonstrated a function for M. tuberculosis SecA2 in suppressing macrophage immune responses, which could explain the role of SecA2 in intracellular growth. Our results provide another example of a relationship between M. tuberculosis virulence and inhibition of the host immune response.     

Hereditary hemochromatosis results in decreased iron acquisition and growth by Mycobacterium tuberculosis within human macrophages.

Iron (Fe) acquisition is essential for the growth of intracellular Mycobacterium tuberculosis (M.tb). How this occurs is poorly understood. Hereditary hemochromatosis is an inherited disease in which most cells become overloaded with Fe. However, hereditary hemochromatosis macrophages have lower than normal levels of intracellular Fe. This suggests M.tb growth should be slower in those cells if macrophage intracellular Fe is used by M.tb. Therefore, we compared trafficking and acquisition of transferrin (Tf)- and lactoferrin (Lf)-chelated Fe by M.tb within the phagosome of monocyte-derived macrophages (MDM) from healthy controls and subjects with hereditary hemochromatosis. M.tb in both sets of macrophages acquired more Fe from Lf than Tf. Fe acquisition by M.tb within hereditary hemochromatosis macrophages was decreased by 84% from Tf and 92% from Lf relative to that in healthy control macrophages. There was no difference in Fe acquired from Tf and Lf by the two macrophage phenotypes. Both acquired 3 times more Fe from Lf than Tf. M.tb infection and incubation with interferon gamma (IFN-gamma) reduced macrophage Fe acquisition by 20% and 50%, respectively. Both Tf and Lf colocalized with M.tb phagosomes to a similar extent, independent of macrophage phenotype. M.tb growth was 50% less in hereditary hemochromatosis macrophages. M.tb growing within macrophages from subjects with hereditary hemochromatosis acquire less Fe compared with healthy controls. This is associated with reduced growth of M.tb. These data support a role for macrophage intracellular Fe as a source for M.tb growth.     

Glutamine synthetase GlnA1 is essential for growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs

To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for L-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of L-glutamine, the effective concentration of L-glutamine in the M. tuberculosis phagosome of THP-1 cells was approximately 10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.     

Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth

Legionella dumoffii is one of the common causes of Legionnaires' disease and is capable of replicating in macrophages. To understand the mechanism of survival within macrophages, transposon mutagenesis was employed to isolate the genes necessary for intracellular growth. We identified four defective mutants after screening 790 transposon insertion mutants. Two transposon insertions were in genes homologous to icmB or dotC, within dot/icm loci, required for intracellular multiplication of L. pneumophila. The third was in a gene whose product is homologous to the 17-kDa antigen forming part of the VirB/VirD4 type IV secretion system of Bartonella henselae. The fourth was in the djlA (for "dnaj-like A") gene. DjlA is a member of the DnaJ/Hsp40 family. Transcomplementation of the djlA mutant restored the parental phenotype in J774 macrophages, A549 human alveolar epithelial cells, and the amoeba Acanthamoeba culbertsoni. Using confocal laser-scanning microscopy and transmission electron microscopy, we revealed that in contrast to the wild-type strain, L. dumoffii djlA mutant-containing phagosomes were unable to inhibit phagosome-lysosome fusion. Transmission electron microscopy also showed that in contrast to the virulent parental strain, the djlA mutant was not able to recruit host cell rough endoplasmic reticulum. Furthermore, the stationary-phase L. dumoffii djlA mutants were more susceptible to H2O2, high osmolarity, high temperature, and low pH than was their parental strain. These results indicate that DjlA is required for intracellular growth and organelle trafficking, as well as bacterial resistance to environmental stress. This is the first report demonstrating that a single DjlA-deficient mutant exhibits a distinct phenotype.     

A genetic screen for Mycobacterium tuberculosis mutants defective for phagosome maturation arrest identifies components of the ESX-1 secretion system

After phagocytosis, the intracellular pathogen Mycobacterium tuberculosis arrests the progression of the nascent phagosome into a phagolysosome, allowing for replication in a compartment that resembles early endosomes. To better understand the molecular mechanisms that govern phagosome maturation arrest, we performed a visual screen on a set of M. tuberculosis mutants specifically attenuated for growth in mice to identify strains that failed to arrest phagosome maturation and trafficked to late phagosomal compartments. We identified 10 such mutants that could be partitioned into two classes based on the kinetics of trafficking. Importantly, four of these mutants harbor mutations in genes that encode components of the ESX-1 secretion system, a pathway critical for M. tuberculosis virulence. Although ESX-1 is required, the known ESX-1 secreted proteins are dispensable for phagosome maturation arrest, suggesting that a novel effector required for phagosome maturation arrest is secreted by ESX-1. Other mutants identified in this screen had mutations in genes involved in lipid synthesis and secretion and in molybdopterin biosynthesis, as well as in genes with unknown functions. Most of these trafficking mutants exhibited a corresponding growth defect during macrophage infection, but two mutants grew like wild-type M. tuberculosis during macrophage infection. Our results support the emerging consensus that multiple factors from M. tuberculosis, including the ESX-1 secretion system, are involved in modulating trafficking within the host.     

Deletion of the Mycobacterium tuberculosis resuscitation-promoting factor Rv1009 gene results in delayed reactivation from chronic tuberculosis

Approximately one-third of the human population is latently infected with Mycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintaining M. tuberculosis in the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five related M. tuberculosis proteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role of M. tuberculosis rpf genes in an established mouse model of M. tuberculosis persistence and reactivation. After an aerosol infection with the M. tuberculosis Erdman wild type (Erdman) or single-deletion rpf mutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the five rpf deletion mutants tested, one (deltaRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and deltaRv1009-infected mice at advanced stages of reactivation. Analysis of rpf gene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the five rpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting that rpf gene expression may be regulated in a growth phase-dependent manner. To our knowledge, deltaRv1009 is the first M. tuberculosis mutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.     

Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival

The Mycobacterium tuberculosis exported repetitive protein (Erp) is a virulence determinant required for growth in cultured macrophages and in vivo. To better understand the role of Erp in Mycobacterium pathogenesis, we generated a mutation in the erp homologue of Mycobacterium marinum, a close genetic relative of M. tuberculosis. erp-deficient M. marinum was growth attenuated in cultured macrophage monolayers and during chronic granulomatous infection of leopard frogs, suggesting that Erp function is similarly required for the virulence of both M. tuberculosis and M. marinum. To pinpoint the step in infection at which Erp is required, we utilized a zebrafish embryo infection model that allows M. marinum infections to be visualized in real-time, comparing the erp-deficient strain to a DeltaRD1 mutant whose stage of attenuation was previously characterized in zebrafish embryos. A detailed microscopic examination of infected embryos revealed that bacteria lacking Erp were compromised very early in infection, failing to grow and/or survive upon phagocytosis by host macrophages. In contrast, DeltaRD1 mutant bacteria grow normally in macrophages but fail to induce host macrophage aggregation and subsequent cell-to-cell spread. Consistent with these in vivo findings, erp-deficient but not RD1-deficient bacteria exhibited permeability defects in vitro, which may be responsible for their specific failure to survive in host macrophages.     

Differential requirements for VirB1 and VirB2 during Brucella abortus infection

The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.     

RpmA is required for nonopsonic phagocytosis of Pseudomonas aeruginosa

Pseudomonas aeruginosa causes severe respiratory tract infections in patients with cystic fibrosis (CF). We have been examining nonopsonic phagocytosis of P. aeruginosa by macrophages. To study the P. aeruginosa-macrophage interaction at the molecular level, we have constructed a transposon Tn5G bank in a clinical isolate of P. aeruginosa (strain 4020) and identified mutants resistant to nonopsonic phagocytosis. Phagocytosis-resistant mutants were enriched by passaging the transposon bank over 18 macrophage monolayers. Of 900 individual mutants isolated from this enriched pool in a nonopsonic phagocytosis assay, we identified 85 putative mutants that were resistant to phagocytosis. In this study, we have characterized one of these transposon mutants, P. aeruginosa 4020 H27A, which was poorly ingested. H27A possessed a Tn5G insertion in a gene encoding a protein with homology to the MotA proteins of several species of bacteria. We have called this gene rpmA for required for phagocytosis by macrophages. RpmA is one of two MotA paralogs in P. aeruginosa. This rpmA::Tn5G mutant was motile both on agar plates and in visual examination of wet mounts. The phagocytosis defect was partially complemented by providing the rpmA gene in trans and fully complemented when both rpmA and rpmB were provided. A rpmA null mutant was ingested by macrophages similar to the H27A transposon mutant. These data suggest that the rpmA and rpmB gene products are required for the efficient ingestion of P. aeruginosa by macrophages.     

Intracellular growth and survival of Salmonella enterica serovar Typhimurium carrying truncated hemoglobins of Mycobacterium tuberculosis

Two distantly related truncated hemoglobins (trHbs), HbN and HbO, are produced at different growth stages of Mycobacterium tuberculosis. Oxygen and nitric oxide (NO) binding properties of these trHbs suggest their vital role(s) in adaptation of tubercle bacillus under hypoxic and nitrosative stress conditions. Here, we have demonstrated that HbN of M. tuberculosis provides distinct advantage over HbO in supporting intracellular growth and survival of the heterologous host, Salmonella enterica serovar Typhimurium, during macrophage infection specifically against toxicity of NO. HbN and HbO encoding genes of M. tuberculosis have been expressed in a NO-sensitive hmp mutant of S. enterica serovar Typhimurium that exhibits attenuated growth within the macrophages. Presence of HbN and HbO conferred distinct oxygen dependent NO metabolizing activity to the mutant S. enterica serovar Typhimurium. However, the HbN carrying cells exhibited nearly 2-3-fold higher NO metabolizing activity than the isogenic strain having HbO under aerobic condition. More than half of the NO uptake activity of HbN carrying cells was retained when oxygen level dropped to microaerobic condition. In comparison, NO uptake activity of HbO carrying cells of mutant S. enterica dropped drastically (90%) under similar hypoxic conditions. When internalized by mice peritoneal macrophages, HbN carrying cells exhibited 3- and 4-fold higher survival compared to similarly bound and internalized HbO carrying and control cells, respectively. The protective effect of HbN persisted even after activation of macrophages in the presence of IFN-gamma, whereas, HbO did not show any significant effect on survival of the NO-sensitive hmp mutant of Salmonella. These results provide strong experimental evidence in support of the protective role of HbN against nitrosative stress inside macrophages and suggest that intracellular protection conferred by HbN of M. tuberculosis might not be restricted to its native host only.     

The M cell as a portal of entry to the lung for the bacterial pathogen Mycobacterium tuberculosis.

M. tuberculosis accesses the terminal lung and is phagocytosed by alveolar macrophages. Utilizing a mouse intratracheal challenge model, we demonstrate that M. tuberculosis rapidly enters through M cells as well. From there, bacilli are deposited within associated intraepithelial leukocytes and subsequently conveyed to the draining lymph nodes early after infection. Osteopetrotic (Csfm(op)/Csfm(op)) mice, null mutants for macrophage colony-stimulating factor, possess diminished numbers of circulating monocytes and tissue macrophages. Csfm(op)/Csfm(op) mice were highly susceptible to challenge with M. tuberculosis. In contrast to controls, tubercle bacilli were not conveyed to draining lymph nodes early after infection but were instead retained within the mucosa. These results indicate that M cells represent an alternate portal of entry for M. tuberculosis, which may contribute to the rapid development of protective lung immune responses.     

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.