Research Article: Type 3 Secretion System Cluster 3 Is a Critical Virulence Determinant for Lung-Specific Melioidosis

Date Published: January 8, 2015

Publisher: Public Library of Science

Author(s): Maria G. Gutierrez, Tia L. Pfeffer, Jonathan M. Warawa, Pamela L. C. Small.

Abstract: Burkholderia pseudomallei, the bacterial agent of melioidosis, causes disease through inhalation of infectious particles, and is classified as a Tier 1 Select Agent. Optical diagnostic imaging has demonstrated that murine respiratory disease models are subject to significant upper respiratory tract (URT) colonization. Because human melioidosis is not associated with URT colonization as a prominent presentation, we hypothesized that lung-specific delivery of B. pseudomallei may enhance our ability to study respiratory melioidosis in mice. We compared intranasal and intubation-mediated intratracheal (IMIT) instillation of bacteria and found that the absence of URT colonization correlates with an increased bacterial pneumonia and systemic disease progression. Comparison of the LD50 of luminescent B. pseudomallei strain, JW280, in intranasal and IMIT challenges of albino C57BL/6J mice identified a significant decrease in the LD50 using IMIT. We subsequently examined the LD50 of both capsular polysaccharide and Type 3 Secretion System cluster 3 (T3SS3) mutants by IMIT challenge of mice and found that the capsule mutant was attenuated 6.8 fold, while the T3SS3 mutant was attenuated 290 fold, demonstrating that T3SS3 is critical to respiratory melioidosis. Our previously reported intranasal challenge studies, which involve significant URT colonization, did not identify a dissemination defect for capsule mutants; however, we now report that capsule mutants exhibit significantly reduced dissemination from the lung following lung-specific instillation, suggesting that capsule mutants are competent to spread from the URT, but not the lung. We also report that a T3SS3 mutant is defective for dissemination following lung-specific delivery, and also exhibits in vivo growth defects in the lung. These findings highlight the T3SS3 as a critical virulence system for respiratory melioidosis, not only in the lung, but also for subsequent spread beyond the lung using a model system uniquely capable to characterize the fate of lung-delivered pathogen.

Partial Text: Burkholderia pseudomallei is the Tier 1 Select Agent bacterial pathogen responsible for the disease melioidosis. B. pseudomallei is found in moist tropical soils worldwide, but has been long characterized to be endemic to Southeast Asia and northern Australia [1]. Naturally acquired disease typically involves percutaneous inoculation or inhalation of pathogen by susceptible hosts, with risk factors including diabetes and alcoholism [2]. Under exceptional conditions, such as natural disasters, otherwise healthy individuals are also susceptible to melioidosis [3], [4], [5], [6], suggesting that dose and route of inoculation are key elements to determining whether or not a healthy individual acquires disease. The ability of B. pseudomallei to establish a lethal respiratory disease, combined with its inherent resistance to numerous classes of antibiotics, highlights the importance of characterizing respiratory melioidosis for the purposes of biodefense. Importantly, no licensed vaccine exists for melioidosis, nor for glanders, which is caused by the very closely related pathogen Burkholderia mallei.

We have previously developed an optical diagnostic imaging model of intranasal respiratory melioidosis and observed that the URT of mice infected in this manner are subject to prominent infection [13]. URT colonization is associated with infection of the nasal-associated lymphoid tissue (NALT) as well as infection of the olfactory bulbs/CNS [7], [15]. As discussed above, descriptions of disease states associated with URT infections have not been described in human melioidosis, and paired analysis of cultures sputum and throat swabs suggests that pneumonia gives rise to presence of B. pseudomallei at the top of the respiratory tract rather than URT carriage seeding a primary infection which descends to the lung [10]. The over-representation of these symptoms in mice have led us, and others, to investigate alternatives to the standard approaches of inoculating mice with B. pseudomallei through the nares. A recently developed intratracheal model of respiratory melioidosis succeeded in abrogating CNS infections, suggesting that URT colonization is directly responsible for the high levels of meningitis reported in the murine model [14], [27]. Our current studies focused on advancing these findings by identifying whether URT infection in the mouse impacts the overall course of disease and ask whether these impacts might influence both basic and translational studies of respiratory melioidosis.



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