Research Article Journal of Innate J Innate Immun 2009;1:494–506 Received: March 3, 2009 Immunity DOI: 10.1159/000225955 Accepted after revision: April 7, 2009 Published online: June 18, 2009 ClpX Contributes to Innate Defense Peptide Resistance and Virulence Phenotypes of Bacillus anthracis a a f b Shauna M. McGillivray Celia M. Ebrahimi Nathan Fisher Mojgan Sabet a g c d Dawn X. Zhang Yahua Chen Nina M. Haste Raffi V. Aroian b b f Richard L. Gallo Donald G. Guiney Arthur M. Friedlander g a, c, e Theresa M. Koehler Victor Nizet a b c Departments of Pediatrics and Medicine, Skaggs School of Pharmacy and Pharmaceutical Sciences, and d e Division of Biological Sciences, University of California San Diego, La Jolla, Calif., Rady Children’s Hospital, f San Diego, Calif. , United States Army Medical Research Institute of Infectious Diseases, Frederick, Md. , and g Department of Microbiology and Molecular Genetics, University of Texas Houston Health Science Center Medical School, Houston, Tex. , USA Key Words tion was linked to degradation of cathelicidin antimicrobial -Antimicrobial peptides ؒ Bacillus anthracis ؒ Bacterial peptides, a front-line effector of innate host defense. B. an infection ؒ Cathelicidins ؒ Hemolysis ؒ Innate immunity ؒ thracis lacking ClpX were rapidly killed by cathelicidin and Protease ؒ Transposon mutagenesis ؒ Virulence factor ␣ -defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirm- Abstract ing cathelicidin to be a critical element of innate defense Bacillus anthracis is a National Institute of Allergy and Infec- against the pathogen. We conclude that ClpX is an impor- tious Diseases Category A priority pathogen and the caus- tant factor allowing B. anthracis to subvert host immune ative agent of the deadly disease anthrax. We applied a clearance mechanisms, and thus represents a novel thera- transposon mutagenesis system to screen for novel chromo- peutic target for prevention or therapy of anthrax, a fore- somally encoded B. anthracis virulence factors. This ap- most biodefense concern. Copyright © 2009 S. Karger AG, Basel proach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and pro- teolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion Introduction of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and mark- Bacillus anthracis is a Gram-positive, spore-forming edly reduced in vivo survival of the fully virulent B. anthracis bacterium and the causative agent of anthrax. Primarily Ames upon intraperitoneal challenge in guinea pigs. The ex- a disease of livestock, anthrax can infect humans through tracellular proteolytic activity dependent upon ClpX func- cutaneous, respiratory or gastrointestinal routes of infec- © 2009 S. Karger AG, Basel Dr. Victor Nizet Department of Pediatrics and Pharmacy and Skaggs School of Fax +41 61 306 12 34 Pharmacy and Pharmaceutical Sciences, University of California E-Mail [email protected] Accessible online at: 9500 Gilman Drive, Mail Code 0687, La Jolla, CA 92093-0687 (USA) www.karger.com www.karger.com/jin Tel. +1 858 534 7408, Fax +1 858 534 5611, E-Mail [email protected] tion. Inhalational anthrax occurs when endospores are ClpX function proved essential for B. anthracis ␤ -he- introduced to the lung and taken up by resident phago- molytic and extracellular proteolytic activities in plate- cytes. Escape of B. anthracis from the phagolysosome based assays. Further analysis of this mutant uncovered into regional lymph nodes leads to extracellular prolif- a role for ClpX in B. anthracis resistance to host antimi- eration and eventually to high bacterial titers in the blood, crobial peptide killing and virulence in several models of shock and death [1] . The high lethality of inhalational systemic anthrax infection. anthrax coupled with extended spore viability makes B. anthracis a paramount biodefense concern. This was il- lustrated in 2001 when a terrorist sent letters containing Materials and Methods anthrax spores to government offices and news organiza- tions in the USA, killing 5 people and infecting 17 oth- Bacterial Strains and Culture Conditions + – + ers. B. anthracis Sterne (pXO1 pXO2 ) and Ames strains (pXO1 pXO2 + ) were propagated in Brain-Heart Infusion (BHI) medium The best characterized virulence factors of B. anthra- (Sigma) or on BHI agar plates under aerobic conditions at 37 ° C. cis are the anthrax toxins, comprised of lethal factor, ede- When antibiotic selection was indicated, the following concentra- ma factor, protective antigen and the antiphagocytic tions were used: 50 ␮ g/ml of kanamycin (Kan), 5 ␮ g/ml of eryth- ␮ ␮ poly-D -glutamate capsule. The structural genes encoding romycin (Em), 3 g/ml of chloramphenicol (Cm) and 1 g/ml of the anthrax toxins and the biosynthetic genes for capsule tetracycline (Tet). synthesis are located on the large plasmids, pXO1 and Construction of B. anthracis Sterne Transposon Mutant pXO2 respectively, and loss of either plasmid attenuates Library B. anthracis virulence in certain animal models [2, 3] . To develop a Himar1 transposon system for use in B. anthracis , While both anthrax toxin and capsule are critical in im- we constructed a delivery plasmid that allowed expression of the mune cell invasion and disease progression, evidence in- Himar1 transposase gene and provided a selectable marker. First, a Himar1-based mini-transposon was constructed by cloning the dicates that chromosome-encoded genes may also con- ⍀ km -2 cassette from pUTE618 [11] into the unique Bgl II site in tribute to B. anthracis pathogenicity [4] . pMMOrf [12] . A hyperactive Himar1 transposase (tnp) gene from To identify novel chromosomal genes involved in vir- pET29C9 [12] was fused to the B. cereus promoter P 4325 [13] . Fi- ulence, we applied a transposon system using the Himar1 nally, the mini- Himar1 cassette and P4325 - Himar1-tnp were cloned transposase to generate a random chromosomal mutant into pUTE583 [11] to generate pUTE664. The Himar-1 transpo- son delivery vector pUTE664 was electroporated into B. anthracis library of B. anthracis Sterne, then screened individual using an established protocol [14] . Electroporation mixtures were mutants for loss of hemolytic activity, a potential marker grown in medium containing Em to select for the transposon de- for bacterial cytotoxins. This strategy yielded a mutant in livery plasmid and allow for chromosomal transposition events. which the transposon insertion mapped to a locus coding Cells were subcultured 6 times without antibiotic selection to fa- for the ClpX ATPase. cilitate loss of the plasmid. Candidate chromosomal transposon insertion mutants were identified as B. anthracis colonies exhibit- Caseinolytic proteases (Clp) are conserved intracellu- ing kanamycin resistance and Em sensitivity. Southern blot anal- lar proteases that eliminate damaged, non-functional ysis of selected colonies indicated a highly random distribution of proteins and also control the lifespan of transcriptional single transposon insertions into the chromosomes of individual regulators and rate-limiting enzymes [5]. The ClpXP pro- mutants. DNA flanking the transposon insertion site was iden- tease is comprised of a proteolytic core, ClpP, that pairs tified by single-primer PCR using outwardly directed primer -؅ -GGGAATCATTTGAAGGTT-3 ؅ , followed by direct DNA se 5 -with the regulatory ATPase ClpX and structurally resem- quencing using the nested primer 5 ؅ -TATGCATTTAATACT . bles the eukaryotic 26s proteosome [6] . ClpX is respon- AGCGACG-3 ؅ sible for recognition, unfolding and cleavage of substrate proteins into large peptides to be fed into the proteolytic Targeted Allelic Replacement Mutagenesis and core. Without ClpX, ClpP is limited to degradation of Complementation Studies Approximately 1,000 bp of sequence immediately upstream of -peptides fewer than 7 amino acids long [7] . ClpX can also clpX was amplified with primers UpClpXFwd 5 ؅ -CATTAGGA function independently of ClpP as a molecular chaperone TCCATACATGCCAATCATTTCAATACC-3 ؅ and UpClpXRev , that assists in protein folding [8] . At least 5 classes of ClpX 5 ؅ -TGGCAGGGCGGGGCGTAATGCGAAGGAGAAACA-3 ؅ recognition signals have been identified. Substrates com- while 1,000 bp of sequence immediately downstream of clpX ؅ prise a diverse array of proteins and include enzymes in- was amplified with primers DnClpXFwd 5 -CCAGTGATTTT TTTCTCCATATTTTCACACCCCTTACA-3 ؅ and DnClpXRev . volved in gene regulation, metabolism and stress response 5 ؅ -CATGACTCGAGCGGAAAAGGCGAAAACTACTCTC-3 ؅ [9] . In Bacillus subtilis , ClpX is required for initiation of The UpClpXRev and DnClpXFwd primers were constructed with sporulation [10] . 25-bp 5؅ extensions corresponding to the 5 ؅ and 3 ؅ ends of the cat ClpX and Anthrax Virulence J Innate Immun 2009;1:494–506 495 gene. The upstream and downstream PCR products were then Sporulation and Germination Assays combined with a 650-bp amplicon of the complete cat gene as Sporulation was assessed in 2 ways. First, spores were visual- templates in a second round of PCR using primers UpClpXFwd ized using phase-contrast microscopy after induction of sporula- and DnClpXRev. The resultant amplicon was sub-cloned into tion for up to 7 days. Second, spore formation was quantified by pHY304, a temperature-sensitive vector, and transformed