Aeromonas spp.-mediated cell-contact cytotoxicity is associated with the presence of type III secretion system

In the study we examined the production of cytotonic and cytotoxic toxins and the presence of a type III secretion system (TTSS) in 64 Aeromonas spp. strains isolated from fecal specimens of patients with gastroenteritis. We observed that contact of the bacteria with host epithelial cells is a prerequisite for their cytotoxicity at 3 h incubation. Cell-contact cytotoxic activity of the strains was strongly associated with the presence of the TTSS. Culture supernatants of the strains induced low cytotoxicity effects at the same time of incubation. Cell-free supernatants of 61 (95%) isolates expressed cytotoxic activity which caused the destruction of HEp-2 cells at 24 h. Moreover, 44% strains were cytotonic towards CHO cells and 46% of strains invaded epithelial cells.     

Proteolytic Activity of Pseudomonas aeruginosa Isolates with TTSS-Mediated Cytotoxicity and Invasiveness to Host Cells

Over 100 of Pseudomonas aeruginosa isolates representing the two TTSS genotypes (exoU ⁻/exoS ⁺ or exoU ⁺/exoS ⁻) were cultured in different media in order to evaluate their proteolytic activities and find a relationship between proteolytic activity and the cytotoxic and/or invasive phenotypes displayed by the strains upon infection of RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial cells (PME). The elastolytic activity, protein concentration, and total proteolytic activity (TPA) were measured in culture supernatants. No significant differences were observed in the median elastolytic activities among cytotoxic/noninvasive, noncytotoxic/invasive, and cytotoxic/invasive phenotypes displayed by P. aeruginosa strains. The only significant difference was noted when isolates of the two different TTSS genotypes were grown in a calcium-depleted minimal medium for induction of TTSS (MI). The exoU ⁻/exoS ⁺ isolates showed significant higher levels of the median elastolytic activity when compared to the exoU ⁺/exoS ⁻ isolates. These two groups of isolates secreted the elastase B (LasB) with distinct molecular masses 158 or 116 kD, respectively. The strains of the two TTSS genotypes secreted similar amount of total proteins; however, the higher values of TPA were observed for the isolates of the exoU ⁺ /exoS ⁻ genotype when grown in MI medium. We concluded that there is no direct relationship between secretion of proteases with elastolytic activity and the cytotoxic and/or invasive phenotypes of the isolates observed upon infection of both RAW 264.7 and PME monolayers. Further studies are needed to find out whether others factors beside proteases could influence the mechanism of host cells intoxication mediated by the P. aeruginosa TTSS-delivered toxins.     

Cytotoxic activity of Serratia marcescens clinical isolates

Twenty Serratia marcescens isolates from clinical specimens were examined for their cytotoxic activity on four cell lines (HEp-2, Vero, CHO, J774). Most of the isolates were found to be cytotoxic to CHO (70%), Vero (75%) and HEp-2 cells (90%). CHO cells were the most sensitive to cell-free supernatants, followed by HEp-2 and Vero cells. Two strains produced cytotonic toxins which caused elongation of CHO cells. Moreover, twelve isolates (60%) revealed cytotoxic potential to macrophage cell line J774. The results indicate that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.     

铜绿假单胞菌临床菌株TTSS表达水平及相关因素分析

Ⅲ型分泌系统（type Ⅲ secretion system, TTSS）是铜绿假单胞菌的重要致病因子．分析临床菌株中TTSS的表达水平,对于研究铜绿假单胞菌的致病机制具有重要意义．本研究通过测定融合报告基因exsA-lacZ和exoT-lacZ编码的β-半乳糖苷酶活力,分析了150株铜绿假单胞菌临床菌株exsA和exoT基因的表达水平,发现71株（47.33%）细菌exsA基因的表达为阳性,65株（43.33%）细菌exoT基因的表达为阳性,基因exsA与exoT表达水平存在正相关（P <0.001）,且不同菌株间两者表达水平差异较大．采用统计学方法对实验结果进一步分析发现,TTSS表达与呼吸道感染等临床症状相关（P <0.05）;与标本的分离时间相关（P =0.029）;与菌株亚胺青霉烯耐药性存在负相关（P <0.05）．本研究结果显示,铜绿假单胞菌临床菌株TTSS表达水平差异较大,与多种因素存在相关性.     

Cytotoxin production in 100 strains of Haemophilus ducreyi from different geographic locations

Abstract One-hundred strains of Haemophilus ducreyi , representing isolates from different parts of the world, including the reference strains, were obtained from different collections and characterized with special reference to cytotoxin production in vitro. The cytotoxic activity on cultured epithelial cells (HEp-2) was examined with two methods. The activity in bacterial sonicates was tested on freshly trypsinated cells and strains manifesting little or no cytotoxic activity in sonicates were investigated using attached living bacteria on HEp-2 cell-monolayers. Sonicates from the majority of the H. ducreyi strains (89%) produced significant cytotoxic effects on HEp-2 cells. The reciprocal cytotoxic titers of the sonicates ranged from 2.4 × 10² to 5.3 × 10⁵. Sonicates of 11 strains had low cytotoxic titers ( 1:3 to 1:81), eight of those originating from Asia and three from Africa. These 11 strains caused no damage to the cell monolayer, indicating that the 11 strains produce little or no cytotoxic activity in vitro. In summary, the majority of H. ducreyi isolates produce cytotoxic activity, which support the hypothesis that the cytotoxin may be an important virulence factor of this species.     

Differentiation in Quinolone Resistance by Virulence Genotype in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading pathogen that has become increasingly resistant to the fluoroquinolone antibiotics due to widespread prescribing. Adverse outcomes have been shown for patients infected with fluoroquinolone-resistant strains. The type III secretion system (TTSS) is a major virulence determinant during acute infections through the injection of effector toxins into host cells. Most strains exhibit a unique TTSS virulence genotype defined by the presence of either exoS or exoU gene encoding two of the effector toxins, ExoS and ExoU, respectively. Specific TTSS effector genotype has been shown previously to differentially impact virulence in pneumonia. In this study, we examined the relationship between TTSS effector genotype and fluoroquinolone resistance mechanisms in a collection of 270 respiratory isolates. We found that a higher proportion of exoU+ strains were fluoroquinolone-resistant compared to exoS+ strains (63% vs 49%, p = 0.03) despite its lower overall prevalence (38% exoU+ vs 56% exoS+). Results from sequencing the quinolone resistance determining regions (QRDRs) of the 4 target genes (gyrA, gyrB, parC, parE) indicated that strains containing the exoU gene were more likely to acquire ≥2 mutations than exoS+ strains at MICs ≤8 µg/ml (13% vs none) and twice as likely to have mutations in both gyrA and parC than exoS+ strains (48% vs 24% p = 0.0439). Our findings indicate that P. aeruginosa strains differentially develop resistance-conferring mutations that correlate with TTSS effector genotype and the more virulent exoU+ subpopulation. Differences in mutational processes by virulence genotype that were observed suggest co-evolution of resistance and virulence traits favoring a more virulent genotype in the quinolone-rich clinical environment.     

Process of Protein Transport by the Type III Secretion System

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.     

Cell death mediated by Vibrio parahaemolyticus type III secretion system 1 is dependent on ERK1/2 MAPK, but independent of caspases

Vibrio parahaemolyticus, which causes gastroenteritis, wound infection, and septicemia, has two sets of type III secretion systems (TTSS), TTSS1 and TTSS2. A TTSS1- deficient vcrD1 mutant of V. parahaemolyticus showed an attenuated cytotoxicity against HEp-2 cells, and a significant reduction in mouse lethality, which were both restored by complementation with the intact vcrD1 gene. V. parahaemolyticus also triggered phosphorylation of mitogenactivated protein kinases (MAPKs) including p38 and ERK1/2 in HEp-2 cells. The ability to activate p38 and ERK1/2 was significantly affected in a TTSS1-deficient vcrD1 mutant. Experiments using MAPK inhibitors showed that p38 and ERK1/2 MAPKs are involved in V. parahaemolyticus-induced death of HEp-2 cells. In addition, caspase-3 and caspase-9 were processed into active forms in V. parahaemolyticus-exposed HEp-2 cells, but activation of caspases was not essential for V. parahaemolyticusinduced death of HEp-2 cells, as shown by both annexin V staining and lactate dehydrogenase release assays. We conclude that secreted protein(s) of TTSS1 play an important role in activation of p38 and ERK1/2 in HEp-2 cells that eventually leads to cell death via a caspaseindependent mechanism.     

Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.     

Process of protein transport by the type III secretion system.

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.     

ExoU is a potent intracellular phospholipase

The combination of a large genome encoding metabolic versatility and conserved secreted virulence determinants makes Pseudomonas aeruginosa a model pathogen that can be used to study host-parasite interactions in many eukaryotic hosts. One of the virulence regulons that likely plays a role in the ability of P. aeruginosa to avoid innate immune clearance in mammals is a type III secretion system (TTSS). Upon cellular contact, the P. aeruginosa TTSS is capable of delivering a combination of at least four different effector proteins, exoenzyme S (ExoS), ExoT, ExoU, and ExoY. Two of the four translocated proteins, ExoS and ExoU, are cytotoxic to cells during infection and transfection. The mechanism of cytotoxicity of ExoS is unclear. ExoU, however, has recently been characterized as a member of the phospholipase A family of enzymes, possessing at least phospholipase A2 activity. Similar to ExoS, ExoT and ExoY, ExoU requires either a eukaryotic-specific modification or cofactor for its activity in vitro. The biologic effects of minimal expression of ExoU in yeast can be visualized by membrane damage to different organelles and fragmentation of the vacuole. In mammalian cells, the direct injection of ExoU causes irreversible damage to cellular membranes and rapid necrotic death. ExoU likely represents a unique enzyme and is the first identified phopholipase virulence factor that is translocated into the cytosol by TTSS.     

Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes disease in a variety of hosts. S. Typhimurium actively invade host cells and typically reside within a membrane-bound compartment called the Salmonella-containing vacuole (SCV). The bacteria modify the fate of the SCV using two independent type III secretion systems (TTSS). TTSS are known to damage eukaryotic cell membranes and S. Typhimurium has been suggested to damage the SCV using its Salmonella pathogenicity island (SPI)-1 encoded TTSS. Here we show that this damage gives rise to an intracellular bacterial population targeted by the autophagy system during in vitro infection. Approximately 20% of intracellular S. Typhimurium colocalized with the autophagy marker GFP-LC3 at 1 h postinfection. Autophagy of S. Typhimurium was dependent upon the SPI-1 TTSS and bacterial protein synthesis. Bacteria targeted by the autophagy system were often associated with ubiquitinated proteins, indicating their exposure to the cytosol. Surprisingly, these bacteria also colocalized with SCV markers. Autophagy-deficient (atg5-/-) cells were more permissive for intracellular growth by S. Typhimurium than normal cells, allowing increased bacterial growth in the cytosol. We propose a model in which the host autophagy system targets bacteria in SCVs damaged by the SPI-1 TTSS. This serves to retain intracellular S. Typhimurium within vacuoles early after infection to protect the cytosol from bacterial colonization. Our findings support a role for autophagy in innate immunity and demonstrate that Salmonella infection is a powerful model to study the autophagy process.     

A genome-wide screen identifies a Bordetella type III secretion effector and candidate effectors in other species

Bordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non-apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome-wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica, the screen identified the first TTSS chaperone-effector locus, btcA-bteA, and we experimentally confirmed its function. Expression of bteA is co-ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica, it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human-adapted subspecies B. pertussis and B. parapertussis. Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.     

Xanthomonas type III effector XopD targets SUMO-conjugated proteins in planta

Xanthomonas campestris pathovar vesicatoria (Xcv) uses the type III secretion system (TTSS) to inject effector proteins into cells of Solanaceous plants during pathogenesis. A number of Xcv TTSS effectors have been identified; however, their function in planta remains elusive. Here, we provide direct evidence for a functional role for a phytopathogenic bacterial TTSS effector in planta by demonstrating that the Xcv effector XopD encodes an active cysteine protease with plant-specific SUMO substrate specificity. XopD is injected into plant cells by the TTSS during Xcv pathogenesis, translocated to subnuclear foci and hydrolyses SUMO-conjugated proteins in vivo. Our studies suggest that XopD mimics endogenous plant SUMO isopeptidases to interfere with the regulation of host proteins during Xcv infection.     

Comparison of Campylobacter isolates from poultry and humans: association between in vitro virulence properties, biotypes, and pulsed-field gel electrophoresis clusters

The in vitro virulence properties of 197 temporally and geographically related Campylobacter isolates from chicken broilers and humans were compared. Comparisons of the virulence properties associated with genotypes and biotypes were made. All isolates adhered to, and 63% invaded, INT-407 cells, whereas 13% were cytotoxic for CHO cells. CHO cell-cytotoxic extracts were also cytotoxic for INT-407 cells, but the sensitivity for Vero cells was variable. The proportion of isolates demonstrating a high invasiveness potential (>1,000 CFU ml(-1)) or Vero cell cytotoxicity was significantly higher for human than for poultry isolates. Invasiveness was associated with Campylobacter jejuni isolates of biotypes 1 and 2, whereas CHO and INT-407 cell cytotoxicity was associated with C. jejuni isolates of biotypes 3 and 4. Cytotoxic isolates were also clustered according to pulsed-field gel electrophoresis profiles.     

The Erwinia chrysanthemi EC16 hrp/hrc gene cluster encodes an active Hrp type III secretion system that is flanked by virulence genes functionally unrelated to the Hrp system

Erwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E. amylovora and Pseudomonas syringae. The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins. DNA sequencing of the complete E. chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E. chrysanthemi TTSS genes were syntenic and similar (>50% amino-acid identity) with their E. amylovora orthologs. However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions. Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates. Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence. Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E. chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors. Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings. Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests. virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids. The E. chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P. syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N. benthamiana leaf cells. However, VirA(1-61)-Cya was not translocated into plant cells, and virA expression was not affected by mutations in E. chrysanthemi Hrp regulator genes hrpL and hrpS. Thus, the 44-kb region of the E. chrysanthemi EC16 genome that is centered on the hrplhrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector.     

Comparison of Campylobacter Isolates from Poultry and Humans: Association between In Vitro Virulence Properties, Biotypes, and Pulsed-Field Gel Electrophoresis Clusters

The in vitro virulence properties of 197 temporally and geographically related Campylobacter isolates from chicken broilers and humans were compared. Comparisons of the virulence properties associated with genotypes and biotypes were made. All isolates adhered to, and 63% invaded, INT-407 cells, whereas 13% were cytotoxic for CHO cells. CHO cell-cytotoxic extracts were also cytotoxic for INT-407 cells, but the sensitivity for Vero cells was variable. The proportion of isolates demonstrating a high invasiveness potential (>1,000 CFU ml⁻¹) or Vero cell cytotoxicity was significantly higher for human than for poultry isolates. Invasiveness was associated with Campylobacter jejuni isolates of biotypes 1 and 2, whereas CHO and INT-407 cell cytotoxicity was associated with C. jejuni isolates of biotypes 3 and 4. Cytotoxic isolates were also clustered according to pulsed-field gel electrophoresis profiles.     

Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94A resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.     

Regulation of the type III secretion system in phytopathogenic bacteria

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.     

Pseudomonas syringae HrpJ is a type III secreted protein that is required for plant pathogenesis, injection of effectors, and secretion of the HrpZ1 Harpin

The bacterial plant pathogen Pseudomonas syringae requires a type III protein secretion system (TTSS) to cause disease. The P. syringae TTSS is encoded by the hrp-hrc gene cluster. One of the genes within this cluster, hrpJ, encodes a protein with weak similarity to YopN, a type III secreted protein from the animal pathogenic Yersinia species. Here, we show that HrpJ is secreted in culture and translocated into plant cells by the P. syringae pv. tomato DC3000 TTSS. A DC3000 hrpJ mutant, UNL140, was greatly reduced in its ability to cause disease symptoms and multiply in Arabidopsis thaliana. UNL140 exhibited a reduced ability to elicit a hypersensitive response (HR) in nonhost tobacco plants. UNL140 was unable to elicit an AvrRpt2- or AvrB1-dependent HR in A. thaliana but maintained its ability to secrete AvrB1 in culture via the TTSS. Additionally, UNL140 was defective in its ability to translocate the effectors AvrPto1, HopB1, and AvrPtoB. Type III secretion assays showed that UNL140 secreted HrpA1 and AvrPto1 but was unable to secrete HrpZ1, a protein that is normally secreted in culture in relatively large amounts, into culture supernatants. Taken together, our data indicate that HrpJ is a type III secreted protein that is important for pathogenicity and the translocation of effectors into plant cells. Based on the failure of UNL140 to secrete HrpZ1, HrpJ may play a role in controlling type III secretion, and in its absence, specific accessory proteins, like HrpZ1, may not be extracellularly localized, resulting in disabled translocation of effectors into plant cells.