The type II secretion system as an underappreciated and understudied mediator of interbacterial antagonism.

Interbacterial antagonism involves all major phyla, occurs across the full range of ecological niches, and has great significance for the environment, clinical arena, and agricultural and industrial sectors. Though the earliest insight into interbacterial antagonism traces back to the discovery of antibiotics, a paradigm shift happened when it was learned that protein secretion systems (e.g., types VI and IV secretion systems) deliver toxic "effectors" against competitors. However, a link between interbacterial antagonism and the Gram-negative type II secretion system (T2SS), which exists in many pathogens and environmental species, is not evident in prior reviews on bacterial competition or T2SS function. A current examination of the literature revealed four examples of a T2SS or one of its known substrates having a bactericidal activity against a Gram-positive target or another Gram-negative. When further studied, the T2SS effectors proved to be peptidases that target the peptidoglycan of the competitor. There are also reports of various bacteriolytic enzymes occurring in the culture supernatants of some other Gram-negative species, and a link between these bactericidal activities and T2SS is suggested. Thus, a T2SS can be a mediator of interbacterial antagonism, and it is possible that many T2SSs have antibacterial outputs. Yet, at present, the T2SS remains relatively understudied for its role in interbacterial competition. Arguably, there is a need to analyze the T2SSs of a broader range of species for their role in interbacterial antagonism. Such investigation offers, among other things, a possible pathway toward developing new antimicrobials for treating disease.

Legionella pneumophila Rhizoferrin Promotes Bacterial Biofilm Formation and Growth within Amoebae and Macrophages.

Previously, we showed that Legionella pneumophila secretes rhizoferrin, a polycarboxylate siderophore that promotes bacterial growth in iron-deplete media and the murine lung. Yet, past studies failed to identify a role for the rhizoferrin biosynthetic gene (lbtA) in L. pneumophila infection of host cells, suggesting the siderophore's importance was solely linked to extracellular survival. To test the possibility that rhizoferrin's relevance to intracellular infection was missed due to functional redundancy with the ferrous iron transport (FeoB) pathway, we characterized a new mutant lacking both lbtA and feoB. This mutant was highly impaired for growth on bacteriological media that were only modestly depleted of iron, confirming that rhizoferrin-mediated ferric iron uptake and FeoB-mediated ferrous iron uptake are critical for iron acquisition. The lbtA feoB mutant, but not its lbtA-containing complement, was also highly defective for biofilm formation on plastic surfaces, demonstrating a new role for the L. pneumophila siderophore in extracellular survival. Finally, the lbtA feoB mutant, but not its complement containing lbtA, proved to be greatly impaired for growth in Acanthamoeba castellanii, Vermamoeba vermiformis, and human U937 cell macrophages, revealing that rhizoferrin does promote intracellular infection by L. pneumophila. Moreover, the application of purified rhizoferrin triggered cytokine production from the U937 cells. Rhizoferrin-associated genes were fully conserved across the many sequenced strains of L. pneumophila examined but were variably present among strains from the other species of Legionella. Outside of Legionella, the closest match to the L. pneumophila rhizoferrin genes was in Aquicella siphonis, another facultative intracellular parasite of amoebae.

Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor.

Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior murine-based modeling indicated that this cytokine response is initiated upon recognition of L. pneumophila by a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Through the use of shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), we determined that TRIF, an adaptor downstream of endosomal TLR3 and TLR4, is required for full cytokine secretion by human primary and cell-line macrophages. By characterizing a further set of TLR KO's in human U937 cells, we discerned that, contrary to the viewpoint garnered from murine-based studies, TLR3 and TLR4 (along with TLR2 and TLR5) are in fact vital to the macrophage response in the early stages of L. pneumophila infection. This conclusion was bolstered by showing that i) chemical inhibitors of TLR3 and TLR4 dampen the cytokine output of primary human macrophages and ii) transfection of TLR3 and TLR4 into HEK cells conferred an ability to sense L. pneumophila. TLR3- and TLR4-dependent cytokines promoted migration of human HL-60 neutrophils across an epithelial layer, pointing to the biological importance for the newfound signaling pathway. The response of U937 cells to L. pneumophila LPS was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), we utilized newly-made KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK are also needed for the response of human macrophages to L. pneumophila. Given the lack of attention given them in the bacterial field, C-type lectin receptors were similarly examined; but, they were not required. Overall, this study arguably represents the most extensive, single-characterization of Legionella-recognition receptors within human macrophages.

Legionella pneumophila Cas2 Promotes the Expression of Small Heat Shock Protein C2 That Is Required for Thermal Tolerance and Optimal Intracellular Infection.

Previously, we demonstrated that Cas2 encoded within the CRISPR-Cas locus of Legionella pneumophila strain 130b promotes the ability of the Legionella pathogen to infect amoebal hosts. Given that L. pneumophila Cas2 has RNase activity, we posited that the cytoplasmic protein is regulating the expression of another Legionella gene(s) that fosters intracellular infection. Proteomics revealed 10 proteins at diminished levels in the cas2 mutant, and reverse transcription-quantitative (qRT-PCR) confirmed the reduced expression of a gene encoding putative small heat shock protein C2 (HspC2), among several others. As predicted, the gene was expressed more highly at 37°C to 50°C than that at 30°C, and an hspC2 mutant, but not its complemented derivative, displayed ~100-fold reduced CFU following heat shock at 55°C. Compatible with the effect of Cas2 on hspC2 expression, strains lacking Cas2 also had impaired thermal tolerance. The hspC2 mutant, like the cas2 mutant before it, was greatly impaired for infection of Acanthamoeba castellanii, a frequent host for legionellae in waters. HspC2 and Cas2 were not required for entry into these host cells but promoted the replicative phase of intracellular infection. Finally, the hspC2 mutant exhibited an additional defect during the infection of macrophages, which are the primary host for legionellae during lung infection. In summary, hspC2 is upregulated by the presence of Cas2, and HspC2 uniquely promotes both L. pneumophila extracellular survival at high temperatures and infection of amoebal and human host cells. To our knowledge, these findings also represent the first genetic proof linking Cas2 to thermotolerance, expanding the repertoire of noncanonical functions associated with CRISPR-Cas proteins.

Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation.

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.

Stenotrophomonas maltophilia Encodes a VirB/VirD4 Type IV Secretion System That Modulates Apoptosis in Human Cells and Promotes Competition against Heterologous Bacteria, Including Pseudomonas aeruginosa.

Stenotrophomonas maltophilia is an emerging opportunistic and nosocomial pathogen. S. maltophilia is also a risk factor for lung exacerbations in cystic fibrosis patients. S. maltophilia attaches to various mammalian cells, and we recently documented that the bacterium encodes a type II secretion system which triggers detachment-induced apoptosis in lung epithelial cells. We have now confirmed that S. maltophilia also encodes a type IVA secretion system (VirB/VirD4 [VirB/D4] T4SS) that is highly conserved among S. maltophilia strains and, looking beyond the Stenotrophomonas genus, is most similar to the T4SS of Xanthomonas To define the role(s) of this T4SS, we constructed a mutant of strain K279a that is devoid of secretion activity due to loss of the VirB10 component. The mutant induced a higher level of apoptosis upon infection of human lung epithelial cells, indicating that a T4SS effector(s) has antiapoptotic activity. However, when we infected human macrophages, the mutant triggered a lower level of apoptosis, implying that the T4SS also elaborates a proapoptotic factor(s). Moreover, when we cocultured K279a with strains of Pseudomonas aeruginosa, the T4SS promoted the growth of S. maltophilia and reduced the numbers of heterologous bacteria, signaling that another effector(s) has antibacterial activity. In all cases, the effect of the T4SS required S. maltophilia contact with its target. Thus, S. maltophilia VirB/D4 T4SS appears to secrete multiple effectors capable of modulating death pathways. That a T4SS can have anti- and prokilling effects on different targets, including both human and bacterial cells, has, to our knowledge, not been seen before.

Type II Secretion Promotes Bacterial Growth within the Legionella-Containing Vacuole in Infected Amoebae.

It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant's defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.

Stenotrophomonas maltophilia Serine Protease StmPr1 Induces Matrilysis, Anoikis, and Protease-Activated Receptor 2 Activation in Human Lung Epithelial Cells.

Stenotrophomonas maltophilia is an emerging, opportunistic nosocomial pathogen that can cause severe disease in immunocompromised individuals. We recently identified the StmPr1 and StmPr2 serine proteases to be the substrates of the Xps type II secretion system in S. maltophilia strain K279a. Here, we report that a third serine protease, StmPr3, is also secreted in an Xps-dependent manner. By constructing a panel of protease mutants in strain K279a, we were able to determine that StmPr3 contributes to the previously described Xps-mediated rounding and detachment of cells of the A549 human lung epithelial cell line as well as the Xps-mediated degradation of fibronectin, fibrinogen, and the cytokine interleukin-8 (IL-8). We also determined that StmPr1, StmPr2, and StmPr3 account for all Xps-mediated effects toward A549 cells and that StmPr1 contributes the most to Xps-mediated activities. Thus, we purified StmPr1 from the S. maltophilia strain K279a culture supernatant and evaluated the protease's activity toward A549 cells. Our analyses revealed that purified StmPr1 behaves more similarly to subtilisin than to trypsin. We also determined that purified StmPr1 likely induces cell rounding and detachment of A549 cells by targeting cell integrin-extracellular matrix connections (matrilysis) as well as adherence and tight junction proteins for degradation. In this study, we also identified anoikis as the mechanism by which StmPr1 induces the death of A549 cells and found that StmPr1 induces A549 IL-8 secretion via activation of protease-activated receptor 2. Altogether, these results suggest that the degradative and cytotoxic activities exhibited by StmPr1 may contribute to S. maltophilia pathogenesis in the lung by inducing tissue damage and inflammation.

Expanding Role of Type II Secretion in Bacterial Pathogenesis and Beyond.

Type II secretion (T2S) is one means by which Gram-negative pathogens secrete proteins into the extracellular milieu and/or host organisms. Based upon recent genome sequencing, it is clear that T2S is largely restricted to the Proteobacteria, occurring in many, but not all, genera in the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria classes. Prominent human and/or animal pathogens that express a T2S system(s) include Acinetobacter baumannii, Burkholderia pseudomallei, Chlamydia trachomatis, Escherichia coli, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Vibrio cholerae, and Yersinia enterocolitica T2S-expressing plant pathogens include Dickeya dadantii, Erwinia amylovora, Pectobacterium carotovorum, Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas oryzae, and Xylella fastidiosa T2S also occurs in nonpathogenic bacteria, facilitating symbioses, among other things. The output of a T2S system can range from only one to dozens of secreted proteins, encompassing a diverse array of toxins, degradative enzymes, and other effectors, including novel proteins. Pathogenic processes mediated by T2S include the death of host cells, degradation of tissue, suppression of innate immunity, adherence to host surfaces, biofilm formation, invasion into and growth within host cells, nutrient assimilation, and alterations in host ion flux. The reach of T2S is perhaps best illustrated by those bacteria that clearly use it for both environmental survival and virulence; e.g., L. pneumophila employs T2S for infection of amoebae, growth within lung cells, dampening of cytokines, and tissue destruction. This minireview provides an update on the types of bacteria that have T2S, the kinds of proteins that are secreted via T2S, and how T2S substrates promote infection.

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages.

Previously, we reported that mutants of Legionella pneumophila lacking a type II secretion (T2S) system elicit higher levels of cytokines (e.g., interleukin-6 [IL-6]) following infection of U937 cells, a human macrophage-like cell line. We now show that this effect of T2S is also manifest upon infection of human THP-1 macrophages and peripheral blood monocytes but does not occur during infection of murine macrophages. Supporting the hypothesis that T2S acts to dampen the triggering of an innate immune response, we observed that the mitogen-activated protein kinase (MAPK) and nuclear transcription factor kappa B (NF-κB) pathways are more highly stimulated upon infection with the T2S mutant than upon infection with the wild type. By using short hairpin RNA to deplete proteins involved in specific pathogen-associated molecular pattern (PAMP) recognition pathways, we determined that the dampening effect of the T2S system was not dependent on nucleotide binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible protein I (RIG-I)-like receptors (RLRs), double-stranded RNA (dsRNA)-dependent protein kinase receptor (PKR), or TIR domain-containing adaptor inducing interferon beta (TRIF) signaling or an apoptosis-associated speck-like protein containing a CARD (ASC)- or caspase-4-dependent inflammasome. However, the dampening effect of T2S on IL-6 production was significantly reduced upon gene knockdown of myeloid differentiation primary response 88 (MyD88), TANK binding kinase 1 (TBK1), or Toll-like receptor 2 (TLR2). These data indicate that the L. pneumophila T2S system dampens the signaling of the TLR2 pathway in infected human macrophages. We also document the importance of PKR, TRIF, and TBK1 in cytokine secretion during L. pneumophila infection of macrophages.

Stenotrophomonas maltophilia produces an EntC-dependent catecholate siderophore that is distinct from enterobactin.

Stenotrophomonas maltophilia, a Gram-negative, multi-drug-resistant bacterium, is increasingly recognized as a key opportunistic pathogen. Thus, we embarked upon an investigation of S. maltophilia iron acquisition. To begin, we determined that the genome of strain K279a is predicted to encode a complete siderophore system, including a biosynthesis pathway, an outer-membrane receptor for ferrisiderophore, and other import and export machinery. Compatible with these data, K279a and other clinical isolates of S. maltophilia secreted a siderophore-like activity when grown at 25-37 °C in low-iron media, as demonstrated by a chrome azurol S assay, which detects iron chelation, and Arnow and Rioux assays, which detect catecholate structures. Importantly, these supernatants rescued the growth of iron-starved S. maltophilia, documenting the presence of a biologically active siderophore. A mutation in one of the predicted biosynthesis genes (entC) abolished production of the siderophore and impaired bacterial growth in low-iron conditions. Inactivation of the putative receptor gene (fepA) prevented the utilization of siderophore-containing supernatants for growth in low-iron conditions. Although the biosynthesis and import loci showed some similarity to those of enterobactin, a well-known catecholate made by enteric bacteria, the siderophore of K279a was unable to rescue the growth of an enterobactin-utilizing indicator strain, and conversely iron-starved S. maltophilia could not use purified enterobactin. Furthermore, the S. maltophilia siderophore displayed patterns of solubility in organic compounds and mobility upon thin-layer chromatography that were distinct from those of enterobactin and its derivative, salmochelin. Together, these data demonstrate that S. maltophilia secretes a novel catecholate siderophore.

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms.

Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages by Legionella pneumophila In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replication between 4 and 8 h postentry and to grow to large numbers in the Legionella-containing vacuole (LCV), as evident at 12 h. At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum (ER)-derived vesicles to LCVs. The mutant did not lose expression or translocation of six type IV secretion effectors (e.g., SidM) that are well known for mediating Rab1B association with the LCV, indicating that T2S promotes the interaction between the LCV and Rab1B via a novel mechanism. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA (shRNA) treatment, indicating that T2S also potentiates events beyond Rab1B association. In support of this, a sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant (and a sidM mutant) and showed a growth difference of as much as a 400-fold compared to the wild type. Together, these data reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes.

Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila.

Legionella pneumophila is an intracellular bacterial pathogen that replicates in alveolar macrophages, causing a severe form of pneumonia. Intracellular growth of the bacterium depends on its ability to sequester iron from the host cell. In the L. pneumophila strain 130b, one mechanism used to acquire this essential nutrient is the siderophore legiobactin. Iron-bound legiobactin is imported by the transport protein LbtU. Here, we describe the role of LbtP, a paralog of LbtU, in iron acquisition in the L. pneumophila strain Philadelphia-1. Similar to LbtU, LbtP is a siderophore transport protein and is required for robust growth under iron-limiting conditions. Despite their similar functions, however, LbtU and LbtP do not contribute equally to iron acquisition. The Philadelphia-1 strain lacking LbtP is more sensitive to iron deprivation in vitro Moreover, LbtP is important for L. pneumophila growth within macrophages while LbtU is dispensable. These results demonstrate that LbtP plays a dominant role over LbtU in iron acquisition. In contrast, loss of both LbtP and LbtU does not impair L. pneumophila growth in the amoebal host Acanthamoeba castellanii, demonstrating a host-specific requirement for the activities of these two transporters in iron acquisition. The growth defect of the ΔlbtP mutant in macrophages is not due to alterations in growth kinetics. Instead, the absence of LbtP limits L. pneumophila replication and causes bacteria to prematurely exit the host cell. These results demonstrate the existence of a preprogrammed exit strategy in response to iron limitation that allows L. pneumophila to abandon the host cell when nutrients are exhausted.

The Legionella pneumophila Siderophore Legiobactin Is a Polycarboxylate That Is Identical in Structure to Rhizoferrin.

Legionella pneumophila, the agent of Legionnaires' disease, secretes a siderophore (legiobactin) that promotes bacterial infection of the lung. In past work, we determined that cytoplasmic LbtA (from Legiobactin gene A) promotes synthesis of legiobactin, inner membrane LbtB aids in export of the siderophore, and outer membrane LbtU and inner membrane LbtC help mediate ferrilegiobactin uptake and assimilation. However, the past studies examined legiobactin contained within bacterial culture supernatants. By utilizing high-pressure liquid chromatography that incorporates hydrophilic interaction-based chemistry, we have now purified legiobactin from supernatants of virulent strain 130b that is suitable for detailed chemical analysis. High-resolution mass spectrometry (MS) revealed that the molecular mass of (protonated) legiobactin is 437.140 Da. On the basis of the results obtained from both MS analysis and various forms of nuclear magnetic resonance, we found that legiobactin is composed of two citric acid residues linked by a putrescine bridge and thus is identical in structure to rhizoferrin, a polycarboxylate-type siderophore made by many fungi and several unrelated bacteria. Both purified legiobactin and rhizoferrin obtained from the fungus Cunninghamella elegans were able to promote Fe(3+) uptake by wild-type L. pneumophila as well as enhance growth of iron-starved bacteria. These results did not occur with 130b mutants lacking lbtU or lbtC, indicating that both endogenously made legiobactin and exogenously derived rhizoferrin are assimilated by L. pneumophila in an LbtU- and LbtC-dependent manner.

Type II Secretion-Dependent Degradative and Cytotoxic Activities Mediated by Stenotrophomonas maltophilia Serine Proteases StmPr1 and StmPr2.

Stenotrophomonas maltophilia is an emerging opportunistic pathogen that primarily causes pneumonia and bacteremia in immunocompromised individuals. We recently reported that S. maltophilia strain K279a encodes the Xps type II secretion system and that Xps promotes rounding, actin rearrangement, detachment, and death in the human lung epithelial cell line A549. Here, we show that Xps-dependent cell rounding and detachment occur with multiple human and murine cell lines and that serine protease inhibitors block Xps-mediated rounding and detachment of A549 cells. Using genetic analysis, we determined that the serine proteases StmPr1 and StmPr2, which were confirmed to be Xps substrates, are predominantly responsible for secreted proteolytic activities exhibited by strain K279a, as well as the morphological and cytotoxic effects on A549 cells. Supernatants from strain K279a also promoted the degradation of type I collagen, fibrinogen, and fibronectin in a predominantly Xps- and protease-dependent manner, although some Xps-independent degradation of fibrinogen was observed. Finally, Xps, and predominantly StmPr1, degraded interleukin 8 (IL-8) secreted by A549 cells during coculture with strain K279a. Our findings indicate that while StmPr1 and StmPr2 are predominantly responsible for A549 cell rounding, extracellular matrix protein degradation, and IL-8 degradation, additional Xps substrates also contribute to these activities. Altogether, our data provide new insight into the virulence potential of the S. maltophilia Xps type II secretion system and its StmPr1 and StmPr2 substrates.

Nuclease activity of Legionella pneumophila Cas2 promotes intracellular infection of amoebal host cells.

Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts.

IroT/mavN, a new iron-regulated gene involved in Legionella pneumophila virulence against amoebae and macrophages.

Legionella pneumophila is a pathogenic bacterium commonly found in water. Eventually, it could be transmitted to humans via inhalation of contaminated aerosols. Iron is known as a key requirement for the growth of L. pneumophila in the environment and within its hosts. Many studies were performed to understand iron utilization by L. pneumophila but no global approaches were conducted. In this study, transcriptomic analyses were performed, comparing gene expression in L. pneumophila in standard versus iron restricted conditions. Among the regulated genes, a newly described one, lpp_2867, was highly induced in iron-restricted conditions. Mutants lacking this gene in L. pneumophila were not affected in siderophore synthesis or utilization. On the contrary, they were defective for growth on iron-depleted solid media and for ferrous iron uptake. A sequence analysis predicts that Lpp_2867 is a membrane protein, suggesting that it is involved in ferrous iron transport. We thus named it IroT, for iron transporter. Infection assays showed that the mutants are highly impaired in intracellular growth within their environmental host Acanthamoeba castellanii and human macrophages. Taken together, our results show that IroT is involved, directly or indirectly, in ferrous iron transport and is a key virulence factor for L. pneumophila.

The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna.

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires' disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts.

Type II secretion and Legionella virulence.

Type II secretion (T2S) is one of six systems that can occur in Gram-negative bacteria for the purpose of secreting proteins into the extracellular milieu and/or into host cells. This chapter will describe the T2S system of Legionella pneumophila. Topics to be covered include the genetic basis of T2S in L. pneumophila, the numbers (>25), types, and novelties of Legionella proteins that are secreted via T2S, and the many ways in which T2S and its substrates promote L. pneumophila physiology, ecology, and virulence. Within the aquatic environment, T2S plays a major role in L. pneumophila intracellular infection of multiple types of (Acanthamoeba, Hartmannella, and Naegleria) amoebae. Within the mammalian host, T2S promotes bacterial persistence in lungs, intracellular infection of both macrophages and epithelial cells, and a dampening of the host innate immune response. In this context, T2S may represent a potential target for both industrial and biomedical application.

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB.

Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).