Bacterial pathogens frequently use protein secretion to mediate interactions with their hosts. Here we found that a virulence locus (HSI-I) of Pseudomonas aeruginosa encodes a protein secretion apparatus. The apparatus assembled in discrete subcellular locations and exported Hcp1, a hexameric protein that forms rings with a 40 angstrom internal diameter. Regulatory patterns of HSI-I suggested that the apparatus functions during chronic infections. We detected Hcp1 in pulmonary secretions of cystic fibrosis (CF) patients and Hcp1-specific antibodies in their sera. Thus, HSI-I likely contributes to the pathogenesis of P. aeruginosa in CF patients. HSI-I-related loci are widely distributed among bacterial pathogens and may play a general role in mediating host interactions.Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the lungs of >80% of cystic fibrosis patients and is the primary cause of morbidity and mortality in these patients (1). A distinguishing feature of the bacterium is its high degree of versatility, which provides P. aeruginosa sufficient phenotypic plasticity to form both acute and chronic infections in humans (2,3). The choice between these disparate life-styles is governed by global virulence regulators, including RetS (regulator of exopolysaccharide and type III secretion) (4) and LadS (lost adherence sensor) (5). RetS and LadS reciprocally regulate virulence determinants such as type III secretion, which is RetS-activated and LadS-repressed, as well as exopolysaccharide production, which is RetS-repressed and LadS-activated. These virulence factors are important in acute and chronic infections, respectively.In addition to characterized virulence pathways, microarray analyses indicated that RetS and LadS reciprocally regulated a functionally uncharacterized virulence locus (Fig. 1). Consistent with its regulatory patterns by RetS and LadS, the virulence locus was required for chronic P. aeruginosa infection of the rat lung ( Fig. 1) homologous to a group of genes found in many Gram-negative proteobacteria that have been termed the IcmF-associated homologous protein (IAHP) cluster (7). P. aeruginosa encodes two other IAHP-related loci elsewhere in its genome; however, these loci were not regulated by either RetS or LadS and have no known role in virulence (Fig. 1). An overview of the distribution and genetic constituents of IAHP loci is shown (table S1).The IAHP-related locus of Vibrio cholerae, which the authors have designated a type VI secretion system, mediates cytotoxicity in phagocytic cells and is required for the extracellular secretion of four proteins lacking canonical hydrophobic amino-terminal signal sequences (8). We postulated that the RetS-and LadS-regulated IAHP locus in P. aeruginosa could play a similar role in extracellular protein targeting. To test this hypothesis, we activated expression of the locus in P. aeruginosa PAO1 by deleting retS. Comparison of the supernatant fractions of ΔretS and wild-type revealed that a small protein (M r ~1...