The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection

Abstract

Burkholderia mallei is a highly pathogenic bacterium that causes the zoonosis glanders. Previous studies indicated that the genome of the organism contains eight genes specifying autotransporter proteins, which are important virulence factors of Gram-negative bacteria. In the present study, we report the characterization of one of these autotransporters, BpaB. Database searches identified the bpaB gene in ten B. mallei isolates and the predicted proteins were 99-100% identical. Comparative sequence analyses indicate that the gene product is a trimeric autotransporter of 1,090 amino acids with a predicted molecular weight of 105-kDa. Consistent with this finding, we discovered that recombinant bacteria expressing bpaB produce a protein of ≥300-kDa on their surface that is reactive with a BpaB-specific monoclonal antibody. Analysis of sera from mice infected with B. mallei indicated that animals produce antibodies against BpaB during the course of disease, thus establishing production of the autotransporter in vivo. To gain insight on its role in virulence, we inactivated the bpaB gene of B. mallei strain ATCC 23344 and determined the median lethal dose of the mutant in a mouse model of aerosol infection. These experiments revealed that the bpaB mutation attenuates virulence 8-14 fold. Using a crystal violet-based assay, we also discovered that constitutive production of BpaB on the surface of B. mallei promotes biofilm formation. To our knowledge, this is the first report of a biofilm factor for this organism.

Antibodies against In Vivo -Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei

“…The plasmids pKASbatA.zeo, pKASilv.kan, pBHR1ΔDra, and pBatA were transferred from E. coli S17 to B. mallei by conjugation as previously outlined (77,92,93,143). All the plasmids were sequenced to ensure that PCR did not introduce mutations that would result in amino acid substitutions in the cloned gene products.…”

“…Specific-pathogen-free (SPF) female BALB/c and C57BL/6 mice were purchased from Envigo and allowed to acclimate for at least 1 week prior to use. After administration of anesthetic, the mice were inoculated intratracheally using a Microsprayer nebulizing device (PennCentury) as previously reported (77,93,144). The infected animals were monitored daily, food and water were provided ad libitum, and humane endpoints were strictly observed.…”

“…For Western blot analyses, equal amounts of protein were resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore), and probed with antibodies as outlined previously (77). Antibody reactivity with protein bands was visualized by chemiluminescence using the substrate Luminata Crescendo Western HRP (EMD Millipore) and a Foto/Analyst Luminary/FX imaging system (Fotodyne Inc.).…”

“…Given their important roles in pathogenicity, overall structure, and cellular location at the host-pathogen interface, autotransporter proteins (ATs) represent excellent targets for developing countermeasures (65)(66)(67)(68)(69)(70)(71)(72)(73)(74). These molecules form one of the largest families of virulence factors in Gram-negative bacteria and contribute a wide range of phenotypes, such as serum resistance, lipolytic activity, biofilms, and host cell adhesion (75)(76)(77)(78)(79). Thus, targeting ATs may impede the ability of pathogenic organisms to establish themselves in a host, persist, and cause disease.…”

“…Previous studies have examined the biological roles of all 8 ATs in B. pseudomallei (88)(89)(90). However, only the oligomeric ATs BimA (intracellular motility protein) (91), BoaA (92) and BpaC (93) (host cell adhesion proteins), and BpaB (biofilm and virulence factor) (77) have been functionally characterized in B. mallei. With this in mind, we sought to characterize the B. mallei ortholog of the conventional AT previously designated BatA in studies with the B. pseudomallei wild-type (WT) isolate K96243 (89) (the strains and plasmids used in the study are listed in Table 1).…”