P. aeruginosa HSI-I is required for secretion of Hcp1 and is active in cystic fibrosis infections. (A) Hcp1 is hypersecreted by ΔretS. SDS–polyacrylamide gel electrophoresis analysis of concentrated culture supernatants from various P. aeruginosa strains. The arrow highlights the position of secreted Hcp1 in ΔretS. (B) Immunoblot analysis of HSI-I–dependent secretion of Hcp1-V. In addition to the genetic alterations indicated, each strain contains hcp1-V. Equal quantities of cell (C) and supernatant (S) fractions were probed with antibodies specific for the β-subunit of RNA polymerase (RNAP) and the VSV-G epitope. (C) Immunoblot analysis of Hcp1 secretion by control strains and a panel of CF patient clinical isolates. (D) Immunoblot analysis of Hcp1 in sputum from CF patients (upper blot). Sputum sample 002-6 is from a CF patient not infected with P. aeruginosa; sputum sample 180-8 is from a CF patient infected with two P. aeruginosa strains that do not secrete Hcp1 (lower blot); and sputum sample 195-1 is from a CF patient infected with a P. aeruginosa strain that actively secretes Hcp1 and a second that does not (lower blot). (E) ELISA analysis of sera from CF patients for antibody response against Hcp1.

Hcp1 forms a hexameric ring with a large internal diameter. (A) Ribbon representation of the Hcp1 monomer colored by secondary structure: β strands, red; α helices, blue; and loops, green. (B) Top view of a ribbon representation of the crystallographic Hcp1 hexamer. The individual subunits are colored differently to highlight their organization. (C) Edge-on view of the Hcp1 hexamer shown in (B). (D) Electron microscopy and single-particle analysis of Hcp1. Electron micrograph of Hcp1 negatively stained with 0.75% (w/v) uranyl formate. Scale bar, 100 nm. (Inset) (Left) Representative class averages and (right) the same averages after six-fold symmetrization. Inset scale bar, 10 nm. (E) Sequence conservation analysis of Hcp1. An alignment of 107 Hcp proteins in 43 Gram-negative bacteria was used to plot the relative degree of conservation at each amino acid on the surface of Hcp1 (see methods in supporting online material). Conservation is indicated by color, where red residues are highly conserved and white residues are poorly conserved.

Overview of P. aeruginosa HSI genes and reciprocal regulation of HSI-I by RetS and LadS. Conserved hypothetical HSI ORFs not discussed in the text (white) and ORFs that lie within the predicted HSI operons (black) are labeled with their genome annotation ORF number [assigned on the basis of (7)]. Predicted paralogous ORFs with prior characterization and those characterized in this study are colored consistently in each locus. The boxed insert shows the position of the hcp2/vgrG2 locus encoded elsewhere in the genome. The boxes beneath HSI-I genes summarize transcriptional profiling data from two prior studies (4, 5). The asterisks denote measurements that meet the statistical significance threshold defined in each study.

ClpV1 is not involved in thermotolerance and localizes to discrete foci in a manner dependent on IcmF and Hcp1. (A) Thermotolerance assay of P. aeruginosa strains bearing a clpB or clpV1 deletion. Cell viability before and after a 25-min heat pulse at 55°C was determined by colony-forming units. The thermotolerance of each strain was normalized relative to wild-type. (B and C) Immunoblot analysis of Hcp1-V secretion by ClpV1 and the AAA-1 mutant ClpV1_E310A (B) and in ΔretS clpV1-gfp (C). (D) Fluorescence microscopy of indicated strains also bearing clpV1-gfp. TMA-DPH is a membrane dye used to highlight the outline of the cells.