title: Peptide nucleic acids inhibit growth of Brucella suis in pure culture and in infected murine macrophages.
abstract: Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar-phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 μM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 μM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.
authors.affiliation: Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
authors.forename: Parthiban
authors.initials: P
authors.lastname: Rajasekaran
authors.forename: Jeffry C
authors.initials: JC
authors.lastname: Alexander
authors.forename: Mohamed N
authors.initials: MN
authors.lastname: Seleem
authors.forename: Neeta
authors.initials: N
authors.lastname: Jain
authors.forename: Nammalwar
authors.initials: N
authors.lastname: Sriranganathan
authors.forename: Alice R
authors.initials: AR
authors.lastname: Wattam
authors.forename: João C
authors.initials: JC
authors.lastname: Setubal
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Boyle
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D041021:Brucella suis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D002006:Brucellosis / Q000382:microbiology*; D002460:Cell Line; D057846:Cell-Penetrating Peptides; D003470:Culture Media; D024881:Drug Resistance, Bacterial; D015870:Gene Expression / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000382:microbiology*; D051379:Mice; D008826:Microbial Sensitivity Tests / Q000379:methods; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D057846:Cell-Penetrating Peptides; D003470:Culture Media; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger