Two systems for targeted gene deletion in Coxiella burnetii

Abstract

Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. C. burnetii's former obligate intracellular nature significantly impeded the genetic characterization of putative virulence factors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. In the present study, we describe two methods for generating targeted gene deletions in C. burnetii that exploit pUC/ColE1 ori-based suicide plasmids encoding sacB for positive selection of mutants. As proof of concept, C. burnetii dotA and dotB, encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre-lox-mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and a loxP site were integrated into 5' and 3' flanking regions of dotA. Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in the deletion of dotA under sucrose counterselection. The second method utilized a loop-in/loop-out strategy to delete dotA and dotB. A single suicide plasmid was first integrated into 5' or 3' target gene flanking regions. Resolution of the plasmid cointegrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. ΔdotA and ΔdotB mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire of C. burnetii genetic tools now allows ready fulfillment of molecular Koch's postulates for suspected virulence genes.

Fig 1

Suicide plasmids used in the present study.

Fig 2

Recovery of C. burnetii Cre-lox-mediated recombinants is enhanced by sacB-based sucrose counterselection. (A) Schematic showing Cre-lox mediated deletion of the 311^P-MC-sacB gene cassette carried by NMII transformed with pMiniTn7T-CAT::311^P-MC-sacB. (B) Agarose gel showing the efficiency of cassette deletion by the NMII-Tn7 transformant in the absence of Cre recombinase, or when cotransformed with a suicide plasmid encoding constitutively expressed cre (pUC19::1169^P-cre), or cre under the control of an aTc-inducible promoter (pUC19::tetRA^P-cre), and grown in the presence or absence of 1% sucrose. An intact Tn7 sequence is indicated by a 3,356-bp PCR product. Deletion of the Tn7-encoded 311^P-MC-sacB gene cassette is indicated by a 639-bp PCR product.

Fig 3

Cre-lox-mediated deletion of dotA. (A) Schematic showing Cre-lox mediated replacement of dotA with a Kan^r gene cassette. The remaining loxP site, BglII (B) and EcoRI (E) restriction sites, and two regions corresponding to Southern blot probe DNA, are shown. (B) Southern blot of BglII/EcoRI-digested genomic DNA from NMII and the NMII/ΔdotA-loxP mutant strain hybridized with two DNA probes specific to regions flanking dotA. Disruption of the dotA-containing 3.9-kb BglII/EcoRI fragment of NMII in the NMII/ΔdotA-loxP mutant strain is indicated by hybridizing 0.7-kb BglII/EcoRI and 1.85-kb EcoRI fragments. (A unique EcoRI site was introduced by the Kan^r gene cassette.) (C) PCR using primers specific to dotA showing the absence of target sequence in the NMII/ΔdotA-loxP mutant strain.

Fig 4

Deletion of dotA and dotB using a single suicide plasmid and a loop-in/loop-out strategy. (A) Schematic showing the replacement of dotA with a Kan^r gene cassette. BglII (B) and EcoRI (E) restriction sites are shown, as are two regions corresponding to probe DNA used in Southern blots. (B) Southern blot of BglII/EcoRI-digested genomic DNA from NMII and the ΔdotA mutant hybridized with two DNA probes specific to regions flanking dotA. Disruption of the dotA-containing 3.9-kb BglII/EcoRI fragment of NMII in the ΔdotA mutant is indicated by a hybridizing 2.5-kb BglII/EcoRI fragment. (C) PCR using primers specific to dotA show the absence of target sequence in the ΔdotA mutant. (D) Schematic showing the replacement of dotB with a Kan^r gene cassette. PstI (P) and SalI (S) restriction sites are shown, as are two regions corresponding to probe DNA used in Southern blots. (E) Southern blot of PstI/SalI-digested genomic DNA from NMII and the ΔdotB mutant hybridized with two DNA probes specific to regions flanking dotB. Disruption of dotB-containing 3.4-kb SalI and 2.0-kb PstI/SalI fragments of NMII in the ΔdotB mutant is indicated by hybridizing 4.1-kb SalI and 1.2-kb PstI/SalI fragments (F) PCR using primers specific to dotB showing the absence of target sequence in the ΔdotB mutant. (G) Immunoblot of ΔdotB mutant lysate showing the absence of the 41.3-kDa DotB protein.

Fig 5

The ΔdotA and ΔdotB mutants are deficient in secretion of T4BSS substrates. Cytosolic levels of cAMP after infection of THP-1 macrophages for 2 days with NMII or Δdot mutants expressing CyaA fused to the previously defined Dot/Icm substrates CpeD and CpeE. Control infections were conducted with NMII expressing CyaA alone. Elevated levels of cAMP, indicating secretion, were observed only with NMII expressing CyaA-CpeD or -CpeE fusion proteins. The results shown are from one experiment conducted in duplicate and representative of two independent experiments.

Fig 6

ΔdotA and ΔdotB mutants are defective in intracellular growth. Six-day genome equivalent (GE) increases of NMII, Δdot mutants, or complemented (comp) Δdot mutants grown in ACCM-2 (A) or Vero cells (B). ACCM-2 results are expressed as the means of three independent experiments. Error bars indicate the standard deviations from the means. Vero cell results are expressed as the means of two biological replicates from three independent experiments. Error bars indicate the standard deviations from the means, and asterisks indicate a statistically significant difference (P < 0.0001) from cells infected with NMII.

Fig 7

ΔdotA and ΔdotB mutants are defective in replication vacuole development. Fluorescence micrographs of Vero cells infected for 4 days with NMII, Δdot mutants, or complemented (comp) Δdot mutants. C. burnetii (red) and LAMP-3 (green) are stained by indirect immunofluorescence. Nuclei (blue) are stained with DAPI. Micrograph insets of cells infected with the ΔdotA or ΔdotB mutant show LAMP-3 (upper inset), C. burnetii (middle inset), and the merged image (lower inset). Mutants were harbored as single organisms in dispersed, tight-fitting LAMP-3-positive vacuoles, while NMII and the complemented mutants were observed in single large and spacious replication vacuoles. Bars, 10 μm.