Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella, we determined that wild type secretes activity against both p-nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70 -75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase (chiA) mutants also had Ϸ75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo. In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.bacterial protein secretion ͉ bacterial virulence ͉ Legionnaires' disease

Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.The gram-negative bacterium Legionella pneumophila is the agent of Legionnaires' disease, a pneumonia which especially affects immunocompromised individuals (28,89). An inhabitant of freshwater environments, L. pneumophila naturally replicates within protozoan hosts and in biofilms (27, 89). Following inhalation of contaminated aerosols, the bacterium reaches the human respiratory tract. Bacterial multiplication in alveolar macrophages is concomitant with cell death and damage to the lung tissue (89, 100).In gram-negative bacteria, the PilD prepilin peptidase is necessary for the cleavage and methylation of pilins and pseudopilins that assemble into type IV pili (Tfp) (55,68,69,87). In addition, PilD processes other pseudopilins that are necessary for the biogenesis of a functional type II protein secretion system (8,13,55,71,86). Accordingly, our previous mutational analysis determined that pilD is required for L. pneumophila piliation and protein secretion (4, 52). In L. pneumophila, Tfp promote attachment to host cells and are involved in competence for DNA transformation (84, 85). The L. pneumophila proteins believed to be secreted via the type II system include a zinc metalloprotease, acid phosphatase...

SummaryThe interaction between the plant pathogen Xanthomonas campestris pv. vesicatoria and its host plants is controlled by hrp genes (hypersensitive reaction and pathogenicity), which encode a type III protein secretion system. Among type III-secreted proteins are avirulence proteins, effectors involved in the induction of plant defence reactions. Using nonpolar mutants, we investigated the role of 12 hrp genes in the secretion of the avirulence protein AvrBs3 from X. c. pv. vesicatoria and a heterologous protein, YopE, from Yersinia pseudotuberculosis. Genes conserved among type III secretion systems (hrcQ, hrcR, hrcS and hrcT) as well as non-conserved genes (hrpB1, hrpB2, hrpB4, hrpB5, hrpD5 and hrpD6) were shown to be required for secretion. Protein localization studies using specific antibodies showed that HrpB1 and HrpB4, as well as the putative ATPase HrcN, were mainly found in the soluble fraction of the bacterial cell. In contrast, HrpB2 and HrpF, which is related to NolX of Rhizobium fredii, are secreted into the culture medium in an hrp-dependent manner. As HrpB2, but not HrpF, is essential for type III protein secretion, there might be a hierarchy in the secretion process. We propose that HrpF, which is dispensable for protein secretion but required for AvrBs3 recognition in planta, functions as a translocator of effector proteins into the host cell.

Studies of essential pathogenicity determinants in Gram-negative bacteria have revealed the conservation of type III protein secretion systems that allow delivery of virulence factors into host cells from plant and animal pathogens. Ten of 21 Hrp proteins of the plant pathogen Xanthomonas campestris pv. vesicatoria have been suggested to be part of a type III machinery. Here, we report the hrp-dependent secretion of two avirulence proteins, AvrBs3 and AvrRxv, by X. campestris pv. vesicatoria strains that constitutively express hrp genes. Secretion occurred without leakage of a cytoplasmic marker in minimal medium containing BSA, at pH 5.4. Secretion was strictly hrp-dependent because a mutant carrying a deletion in hrcV, a conserved hrp gene, did not secrete AvrBs3 and AvrRxv. Moreover, the Hrp system of X. campestris pv. vesicatoria was able to secrete proteins from two other plant pathogens: PopA, a protein secreted via the Hrp system in Ralstonia solanacearum, and AvrB, an avirulence protein from Pseudomonas syringae pv. glycinea. Interestingly, X. campestris pv. vesicatoria also secreted YopE, a type IIIsecreted cytotoxin of the mammalian pathogen Yersinia pseudotuberculosis in a hrp-dependent manner. YerA, a YopEspecific chaperone, was required for YopE stability but not for secretion in X. campestris pv. vesicatoria. Our results demonstrate the functional conservation of the type III system of X. campestris for secretion of proteins from both plant and mammalian pathogens and imply recognition of their respective secretion signals.Studies of bacterial pathogens of plants and mammals have revealed common strategies to interact with their hosts. A striking example is the conservation of a protein-secretion system essential for pathogenicity in distantly related Gramnegative bacteria. Type III secretion systems serve to deliver virulence factors into host cells and have been identified in 10 pathogenic bacteria, including the mammalian pathogens Yersinia spp., Shigella flexneri, Salmonella spp., enteropathogenic Escherichia coli, Pseudomonas aeruginosa, and Chlamydia spp., and the plant pathogens Erwinia spp., Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas campestris (for a review on type III systems, see ref.

Previously, we had demonstrated that a Legionella pneumophila prepilin peptidase (pilD) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (pilE L ) mutant. To determine whether these pilD-dependent defects were linked to type II secretion, we have constructed two new mutants of L. pneumophila strain 130b. Mutations were introduced into either lspDE, which encodes the type II outer membrane secretin and ATPase, or lspFGHIJK, which encodes the pseudopilins. Unlike the wild-type and pilE L strains, both lspDE and lspG mutants showed reduced secretion of six pilD-dependent enzymatic activities; i.e., protease, acid phosphatase, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of the pilD mutant, suggesting that their surfaces are distinct. The pilD, lspDE, and lspG mutants were similarly and greatly impaired for growth within Hartmannella vermiformis, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, both lsp mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, the pilD mutant was clearly 100-fold more defective than the type II secretion mutants in U937 cells. These results suggest the existence of a novel pilD-dependent mechanism for promoting L. pneumophila intracellular infection of human cells.The gram-negative bacterium Legionella pneumophila is the agent of Legionnaires' disease, a potentially fatal pneumonia (14,66). In nature, the organism replicates within protozoan hosts and biofilms found in aquatic environments (8,16,24). Following inhalation of aerosolized droplets, L. pneumophila invades and multiplies within alveolar macrophages (1,57,60,64,66). Various factors that are associated with L. pneumophila infection of protozoa and macrophages have been reported. These include major outer membrane proteins (29, 37), Mip (18,27), flagella (49), type IV pili (58), a catalaseperoxidase (12), growth phase (17), iron acquisition (63), and the Dot-Icm putative type IV secretion apparatus (52,55,64).Our previous studies have shown that a prepilin peptidase gene, pilD, is essential for Legionella growth in amoebae and human macrophages (38, 39). Moreover, a pilD mutant is dramatically reduced in virulence, following intratracheal inoculation of guinea pigs (38). By virtue of their ability to process pilin and the so-called pseudopilins, prepilin peptidases are implicated both in the formation of type IV pili and in protein secretion (41,45,46,48,59). One set of pseudopilins is involved in the assembly of the pili, and another one is involved in the genesis of a functional type II protein secretion system....