Intracellular Growth Defect Research Articles

Cryptococcal phospholipase B1 is required for intracellular proliferation and control of titan cell morphology during macrophage infection.

Cryptococcus neoformans is an opportunistic fungal pathogen and a leading cause of fungal-infection-related fatalities, especially in immunocompromised hosts. Several virulence factors are known to play a major role in the pathogenesis of cryptococcal infections, including the enzyme phospholipase B1 (Plb1). Compared to other well-studied Cryptococcus neoformans virulence factors such as the polysaccharide capsule and melanin production, very little is known about the contribution of Plb1 to cryptococcal virulence. Phospholipase B1 is a phospholipid-modifying enzyme that has been implicated in multiple stages of cryptococcal pathogenesis, including initiation and persistence of pulmonary infection and dissemination to the central nervous system, but the underlying reason for these phenotypes remains unknown. Here we demonstrate that a Δplb1 knockout strain of C. neoformans has a profound defect in intracellular growth within host macrophages. This defect is due to a combination of a 50% decrease in proliferation and a 2-fold increase in cryptococcal killing within the phagosome. In addition, we show for the first time that the Δplb1 strain undergoes a morphological change during in vitro and in vivo intracellular infection, resulting in a subpopulation of very large titan cells, which may arise as a result of the attenuated mutant's inability to cope within the macrophage.

A Type IV Translocated Legionella Cysteine Phytase Counteracts Intracellular Growth Restriction by Phytate

The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.

Importance of branched-chain amino acid utilization in Francisella intracellular adaptation.

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.

Metabolism of the vacuolar pathogen Legionella and implications for virulence.

Legionella pneumophila is a ubiquitous environmental bacterium that thrives in fresh water habitats, either as planktonic form or as part of biofilms. The bacteria also grow intracellularly in free-living protozoa as well as in mammalian alveolar macrophages, thus triggering a potentially fatal pneumonia called “Legionnaires' disease.” To establish its intracellular niche termed the “Legionella-containing vacuole” (LCV), L. pneumophila employs a type IV secretion system and translocates ~300 different “effector” proteins into host cells. The pathogen switches between two distinct forms to grow in its extra- or intracellular niches: transmissive bacteria are virulent for phagocytes, and replicative bacteria multiply within their hosts. The switch between these forms is regulated by different metabolic cues that signal conditions favorable for replication or transmission, respectively, causing a tight link between metabolism and virulence of the bacteria. Amino acids represent the prime carbon and energy source of extra- or intracellularly growing L. pneumophila. Yet, the genome sequences of several Legionella spp. as well as transcriptome and proteome data and metabolism studies indicate that the bacteria possess broad catabolic capacities and also utilize carbohydrates such as glucose. Accordingly, L. pneumophila mutant strains lacking catabolic genes show intracellular growth defects, and thus, intracellular metabolism and virulence of the pathogen are intimately connected. In this review we will summarize recent findings on the extra- and intracellular metabolism of L. pneumophila using genetic, biochemical and cellular microbial approaches. Recent progress in this field sheds light on the complex interplay between metabolism, differentiation and virulence of the pathogen.

A screen of Coxiella burnetii mutants reveals important roles for Dot/Icm effectors and host autophagy in vacuole biogenesis.

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.

Essential Role for the Response Regulator PmrA in Coxiella burnetii Type 4B Secretion and Colonization of Mammalian Host Cells

Successful host cell colonization by the Q fever pathogen, Coxiella burnetii, requires translocation of effector proteins into the host cytosol by a Dot/Icm type 4B secretion system (T4BSS). In Legionella pneumophila, the two-component system (TCS) PmrAB regulates the Dot/Icm T4BSS and several additional physiological processes associated with pathogenesis. Because PmrA consensus regulatory elements are associated with some dot/icm and substrate genes, a similar role for PmrA in regulation of the C. burnetii T4BSS has been proposed. Here, we constructed a C. burnetii pmrA deletion mutant to directly probe PmrA-mediated gene regulation. Compared to wild-type bacteria, C. burnetii ΔpmrA exhibited severe intracellular growth defects that coincided with failed secretion of effector proteins. Luciferase gene reporter assays demonstrated PmrA-dependent expression of 5 of 7 dot/icm operons and 9 of 11 effector-encoding genes with a predicted upstream PmrA regulatory element. Mutational analysis verified consensus sequence nucleotides required for PmrA-directed transcription. RNA sequencing and whole bacterial cell mass spectrometry of wild-type C. burnetii and the ΔpmrA mutant uncovered new components of the PmrA regulon, including several genes lacking PmrA motifs that encoded Dot/Icm substrates. Collectively, our results indicate that the PmrAB TCS is a critical virulence factor that regulates C. burnetii Dot/Icm secretion. The presence of PmrA-responsive genes lacking PmrA regulatory elements also suggests that the PmrAB TCS controls expression of regulatory systems associated with the production of additional C. burnetii proteins involved in host cell parasitism.

A Method for Functional Trans-Complementation of Intracellular Francisella tularensis

Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional trans-complementation using bead-bound bacteria co-infections is a method to rapidly identify mutants that fail to modify a host response. Francisella tularensis is a facultative intracellular bacterial pathogen and is the causative agent of the disease tularemia. F. tularensis enters host cells through phagocytosis, escapes the phagosome, and replicates in the host cell cytosol while suppressing cytokine secretion [1]–[4]. Although substantial progress has been made in understanding the intracellular life cycle of F. tularensis, the F. tularensis proteins responsible for manipulating many host cell pathways are unknown. Identifying novel host-pathogen effector proteins is difficult because there is no rapid method to reliably distinguish between bacterial proteins that modify host processes and proteins that are involved in bacterial processes that are required for the bacteria to survive or replicate in the intracellular environment. The ability to identify mutants that are deficient for host-pathogen interactions is important because it can aid in prioritizing the investigation of genes of interest and in downstream experimental design. Moreover, certain mutant phenotypes, such as decreased phagosomal escape, hinder investigation of other potential phenotypes. A method to specifically complement these phenotypes would allow for further characterizations of certain F. tularensis mutants. Thus we sought to develop a method to easily identify and functionally complement mutants that are deficient for interactions with the host.

The progeny ofLegionella pneumophilain human macrophages shows unique developmental traits

The Gram-negative bacterium Legionella pneumophila is an intracellular parasite of amoebae and an accidental human pathogen that causes a noncommunicable atypical pneumonia known as Legionnaires' disease (LD). In some mammalian cells (e.g. HeLa), L.pneumophila follows a biphasic developmental cycle, differentiating between a replicative form that actively multiplies intracellularly, and a mature infectious form (MIF) that emerges as progeny. To date, it is not known whether the L.pneumophila progenies that emerge from amoebae and human macrophages reach similar developmental stages. Here, we demonstrate that in relation to the fully differentiated and highly infectious MIFs that emerge from amoebae, the L.pneumophila progeny that emerges from macrophages is morphologically undifferentiated, less resistant to antibiotics and less able to initiate infections. However, the L.pneumophila progeny from macrophages did not show any defects in intracellular growth. We thus concluded that macrophage infection with L.pneumophila yields a low number of bona fide MIFs. Because MIFs are the transmissive forms of L.pneumophila produced in vivo, our results showing that they are not efficiently produced in cultured macrophages provide an initial insight into why LD is not communicable.

Asparagine assimilation is critical for intracellular replication and dissemination ofFrancisella

In order to develop a successful infectious cycle, intracellular bacterial pathogens must be able to adapt their metabolism to optimally utilize the nutrients available in the cellular compartments and tissues where they reside. Francisella tularensis, the agent of the zoonotic disease tularaemia, is a highly infectious bacterium for a large number of animal species. This bacterium replicates in its mammalian hosts mainly in the cytosol of infected macrophages. We report here the identification of a novel amino acid transporter of the major facilitator superfamily of secondary transporters that is required for bacterial intracellular multiplication and systemic dissemination. We show that inactivation of this transporter does not affect phagosomal escape but prevents multiplication in the cytosol of all cell types tested. Remarkably, the intracellular growth defect of the mutant was fully and specifically reversed by addition of asparagine or asparagine-containing dipeptides as well as by simultaneous addition of aspartic acid and ammonium. Importantly, bacterial virulence was also restored in vivo, in the mouse model, by asparagine supplementation. This work unravels thus, for the first time, the importance of asparagine for cytosolicmultiplication of Francisella. Amino acid transporters are likely to constitute underappreciated players in bacterial intracellular parasitism.

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.

Identification of Coxiella burnetii Type IV Secretion Substrates Required for Intracellular Replication and Coxiella-Containing Vacuole Formation

Coxiella burnetii, the etiological agent of acute and chronic Q fever in humans, is a naturally intracellular pathogen that directs the formation of an acidic Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Central to its pathogenesis is a specialized type IVB secretion system (T4SS) that delivers effectors essential for intracellular replication and CCV formation. Using a bioinformatics-guided approach, 234 T4SS candidate substrates were identified. Expression of each candidate as a TEM-1 β-lactamase fusion protein led to the identification of 53 substrates that were translocated in a Dot/Icm-dependent manner. Ectopic expression in HeLa cells revealed that these substrates trafficked to distinct subcellular sites, including the endoplasmic reticulum, mitochondrion, and nucleus. Expression in Saccharomyces cerevisiae identified several substrates that were capable of interfering with yeast growth, suggesting that these substrates target crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Among these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells but displayed normal growth in bacteriological medium. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation and suggest less redundancy in effector function than has been found in the comparative Legionella Dot/Icm model.

A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication

Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems.

Altered host immune responses, not lowered growth, are the mechanism of attenuation of Francisella tularensis clpB (P3126)

Abstract Bacterial infections typically initiate both innate and adaptive immune responses. Bacterial attenuation is thought of as altered bacterial growth in the presence of constant immune pressure. In vivo screens have identified F. tularensis genes necessary for virulence. While many of these mutations render F. tularensis incapable of intracellular growth, some mutations have no impact on intracellular growth. We hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS mutant clpB (FTL_0094). Our LVS clpB strain is attenuated in pneumonic tularemia yet we did not observe an intracellular growth defect in bone marrow derived macrophages. We showed that LVS clpB induced an altered innate, but equivalent adaptive, immune response compared to LVS. In B6 mice, LVS clpB induced pro-inflammatory cytokine production in the lung early after infection in contrast to wild-type LVS infection. LVS clpB provoked a robust protective adaptive immune response similar in magnitude to LVS, but with increased IFN-γ and IL-17A production in the lung as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of non-essential gene(s) that cause attenuation, like F. tularensis clpB, can help elucidate bacterial immune evasion mechanisms.

Infection with Francisella tularensis LVSclpBLeads to an Altered yet Protective Immune Response

Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.

Nutritional virulence of Francisella tularensis

Nutritional virulence of Francisella tularensis

Targeting nutrient retrieval by Francisella tularensis

Targeting nutrient retrieval by Francisella tularensis

Disruption of Francisella tularensis Schu S4 iglI , iglJ , and pdpC Genes Results in Attenuation for Growth in Human Macrophages and In Vivo Virulence in Mice and Reveals a Unique Phenotype for pdpC

Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10(8) CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.

TetR-Based Gene Regulation Systems for Francisella tularensis

There are a number of genetic tools available for studying Francisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems for F. tularensis based on the Tet repressor, TetR. For the inducible system, a tet operator sequence was cloned into a modified F. tularensis groESL promoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon, luxCDABE, was cloned under the hybrid Francisella tetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association with tetO. Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml(-1). In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists of FTRp regulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with the lux reporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploring F. tularensis protein function.

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.

Minimization of the Legionella pneumophila genome reveals chromosomal regions involved in host range expansion

Legionella pneumophila is a bacterial pathogen of amoebae and humans. Intracellular growth requires a type IVB secretion system that translocates at least 200 different proteins into host cells. To distinguish between proteins necessary for growth in culture and those specifically required for intracellular replication, a screen was performed to identify genes necessary for optimal growth in nutrient-rich medium. Mapping of these genes revealed that the L. pneumophila chromosome has a modular architecture consisting of several large genomic islands that are dispensable for growth in bacteriological culture. Strains lacking six of these regions, and thus 18.5% of the genome, were viable but required secondary point mutations for optimal growth. The simultaneous deletion of five of these genomic loci had no adverse effect on growth of the bacterium in nutrient-rich media. Remarkably, this minimal genome strain, which lacked 31% of the known substrates of the type IVB system, caused only marginal defects in intracellular growth within mouse macrophages. In contrast, deletion of single regions reduced growth within amoebae. The importance of individual islands, however, differed among amoebal species. The host-specific requirements of these genomic islands support a model in which the acquisition of foreign DNA has broadened the L. pneumophila host range.