An in vivo gene deletion system for determining temporal requirement of bacterial virulence factors

Abstract

Analysis of phenotypes associated with specific mutants has been instrumental in determining the roles of a bacterial gene in a biological process. However, this technique does not allow one to address whether a specific gene or gene set is necessary to maintain such a process once it has been established. In the study of microbial pathogenesis, it is important but difficult to determine the temporal requirement of essential pathogenic determinants in the entire infection cycle. Here we report a Cre/loxP-based genetic system that allowed inducible deletion of specific bacterial genes after the pathogen had been phagocytosed by host cells. Using this system, we have examined the temporal requirement of the Dot/Icm type IV protein transporter of Legionella pneumophila during infection. We found that deletion of single essential dot/icm genes did not prevent the internalized bacteria from completing one cycle of intracellular replication. Further analyses indicate that the observed phenotypes were due to the high stability of the examined Dot/Icm protein. However, postinfection deletion within 8 h of the gene coding for the Dot/Icm substrate, SdhA, abolishes intracellular bacterial growth. This result indicates that the Dot/Icm transporter is important for intracellular bacterial growth after the initial biogenesis of the vacuole. Our study has provided a technical concept for analyzing the temporal requirement of specific bacterial proteins or protein complexes in infection or development.

Fig. 1.

Schematic diagram of a chromosome encoded system for IPTG-inducible deletion of bacterial genes. (A) Structure of the intergenic region between lpg2528 (a hypothetical protein) and lpg2529 (a putative α-amylase), into which the floxed gene is inserted. (B) An oriR6K-based plasmid used for introduction of floxed bacterial genes into corresponding deletion mutants. This plasmid contains a multiple cloning site and a kanamycin resistance gene flanked by two directly repeated loxP sites flanked by two DNA fragments upstream (black bar) and downstream (white bar) of the insertion site. Promoter (arrow P) of the floxed gene was cloned upstream of the first loxP site. The sacB and Cm resistance genes were used to select for recombinant strains that harbor the floxed gene correctly inserted at this chromosomal locus. (C) A plasmid harboring cre that is tightly controlled by LacI^Q. Multiple copies of the lac operator sequence (lacO) situated downstream of the Ptrc promoter were used to achieve controlled expression of cre. The thyA gene conferring thymidine autotrophic to the mutant was used to maintain the plasmid in L. pneumophila. (D) Deletion of the floxed icmQ gene in bacteria grown in broth. Bacterial cultures of OD₆₀₀ = 3.0 were split into two subcultures, and IPTG was added to one of them. At the indicated time points, a fraction of each culture was withdrawn, washed with PBS, and plated onto nonselective media and media containing kanamycin, respectively. (E) IPTG-induced deletion of floxed icmQ in intracellular bacteria. One hour after uptake, infections were induced for 2 h. After removing the inducer, bacterial counts were enumerated to obtain the ratios of kanamycin-resistant cells. Similar results were obtained in several independent experiments.

Fig. 2.

Intracellular growth and levels of IcmQ protein after IPTG-induced gene deletion. (A) Deletion of icmQ in intracellular bacteria did not abolish the first cycle of replication. Mouse macrophages were infected with indicated strains at an MOI of 0.05 for 1 h. IPTG was then added for 5 h to a subset of samples infected with the icmQ knock-in strain. Bacterial counts at the indicated time points were determined. Data shown were from a representative of several independent experiments performed in triplicate. (B) IcmQ levels in broth-grown bacteria after gene deletion. IPTG was added to bacterial cultures at the indicated cell densities, and samples withdrawn at the indicated time points were probed for IcmQ. The cytosolic isocitrate dehydrogenase (ICDH) was probed as a loading control. (C) IcmQ levels in bacteria grown in macrophages. Lysates prepared from cells infected for 24 h were subjected to immunoprecipitation with an anti-IcmQ antibody, and SDS/PAGE-resolved samples were probed. Note that the ≈100-kDa band nonspecifically recognized by the antibody was used as a loading control.

Fig. 3.

The Dot/Icm transporter remained active after IPTG-induced icmQ gene deletion. (A) Deletion of icmQ followed by 10 h of Cm treatment did not abolish bacterial intracellular replication. Infections were conducted as in Fig. 2. After removing IPTG, infected cells were incubated with 5 μg/ml Cm for 10 h and the bacterial growth were allowed to continue for 19 h. (B) Association of calnexin with bacterial vacuoles. Postnucleus supernatant was prepared from infected cells at the indicated times and was stained with a calnexin-specific antibody. The 16-h point includes 5 h of IPTG induction followed by 10 h of Cm treatment. The 26-h point includes 10 h of growth after removing Cm. (C and D) Translocation of LidA. Cells infected at an MOI of 5 were stained and scored for LidA (C) or were fractionated with digitonin followed by immunobloting (D). Hsp70 on the same membrane was probed as a loading control. Similar results were obtained in at least three independent experiments, and data shown were from one representative experiment. (E) IcmQ levels in infected cells after Cm treatment and recovery. Lysates of infected cells were subjected to immunoprecipitation with an anti-IcmQ antibody, and the protein was detected by Western blot. Note that the nonspecific band of ≈100 kDa was used as a loading control.

Fig. 4.

Deletion of sdhA within 8 h after phagocytosis abolished bacterial intracellular growth. (A) Growth of L. pneumophila after IPTG-induced sdhA deletion. Macrophages were infected at an MOI of 0.05, and the inducer was added at the indicated times. Growth of the bacteria was determined 24 h after uptake. (B) Kinetics of the growth after sdhA deletion. The growth of the bacteria in infections similar to A was determined at 4-h time intervals. Similar results were obtained in three independent experiments each done in triplicate. (C) Detection of SdhA after gene deletion. Protein in the soluble fraction of lysates of infected cells was obtained by TCA precipitation, and SdhA was detected by immunoblotting. The Hsp70 was probed as a loading control.

Fig. 5.

Deletion of sdhA during infection led to cell death. (A) Extent of cell death caused by the sdhA mutation and by in vivo gene deletion. Macrophages infected at an MOI of 1 were withdrawn at various time points after phagocytosis, and cell death was assessed by TUNEL staining. (B) Deletion of sdhA in replicating bacteria caused less cell death. IPTG was added to infections at the indicated times after phagocytosis; samples were processed for TUNEL staining after an additional 8 h of incubation.