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Abstract
Burkholderia mallei (Bm) and Burkholderia pseudomallei (Bp) are two closely-related Gram-negative bacteria that cause the severe respiratory illnesses glanders and melioidosis, respectively. Both diseases occur as an acute or chronic bacterial pneumonia. They cause high mortality, are resistant to most antibiotics, and have no vaccine available. Additionally, there are concerns that they could be misused as biological warfare agents, leading to their classification as Tier 1 Select Agents. Being highly related, the feasibility of devising a single vaccine protecting against both organisms seems high. Additionally, we have shown that protective antibodies generated during infection with Bm are capable of conferring protection against both organisms.
Many candidates have been identified, but there remains a need to discover novel immunoprotective antigens as current lead targets fail to prevent chronic infection. One major class of targets is autotransporter proteins. Autotransporters form one of the largest families of virulence factors in Gram-negative bacteria and cause a wide range of pathogenic phenotypes. They are expressed at the bacterial surface which puts them at the host-pathogen interface. While characterizing several autotransporters we found several were not produced in vitro, but were expressed in vivo evidenced by the production of antibodies against the proteins during Bm infection in mice.
Here, we discuss the use of protective antibodies from survivor mice infected with Bm to probe the NEB PhD-12 phage display library to find mimetopes reactive with antibodies which were used to discover novel in vivo expressed Bm antigens. After determining the amino acid sequence of the mimetope, we probed the proteome of Bm and found proteins containing the same sequence within their structure. Additionally, this dissertation discusses the characterization of an in vivo expressed peptidoglycan associated lipoprotein (pal) found in both Bm and Bp. Pal was shown to be a component of the B. mallei cell wall and disrupting its function removed the ability of B. mallei to survive and persist. Finally, we discuss the involvement of BorP in the in vivo expression of the high value target antigen BpaB. BorP is an OmpR-like DNA-binding response regulator that modulates the in vivo expression of BpaB.
Many candidates have been identified, but there remains a need to discover novel immunoprotective antigens as current lead targets fail to prevent chronic infection. One major class of targets is autotransporter proteins. Autotransporters form one of the largest families of virulence factors in Gram-negative bacteria and cause a wide range of pathogenic phenotypes. They are expressed at the bacterial surface which puts them at the host-pathogen interface. While characterizing several autotransporters we found several were not produced in vitro, but were expressed in vivo evidenced by the production of antibodies against the proteins during Bm infection in mice.
Here, we discuss the use of protective antibodies from survivor mice infected with Bm to probe the NEB PhD-12 phage display library to find mimetopes reactive with antibodies which were used to discover novel in vivo expressed Bm antigens. After determining the amino acid sequence of the mimetope, we probed the proteome of Bm and found proteins containing the same sequence within their structure. Additionally, this dissertation discusses the characterization of an in vivo expressed peptidoglycan associated lipoprotein (pal) found in both Bm and Bp. Pal was shown to be a component of the B. mallei cell wall and disrupting its function removed the ability of B. mallei to survive and persist. Finally, we discuss the involvement of BorP in the in vivo expression of the high value target antigen BpaB. BorP is an OmpR-like DNA-binding response regulator that modulates the in vivo expression of BpaB.