The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis

Abstract

BackgroundBurkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC). Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50) is typically ≥ 104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species.ResultsMadagascar hissing cockroaches (MH cockroaches) possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was <10 colony-forming units (cfu) for all three species. In comparison, the LD50 for Escherichia coli in MH cockroaches was >105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1) mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs) with intracellular B. pseudomallei, which indicates that infected hemocytes can fuse while flowing through the insect’s open circulatory system in vivo.ConclusionsThe results demonstrate that MH cockroaches are an attractive alternative to mammals to study host-pathogen interactions and may allow the identification of new Burkholderia virulence determinants. The importance of T6SS-1 as a virulence factor in MH cockroaches and rodents suggests that the primary role of this secretion system is to target evasion of the innate immune system.

pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells

“…Antimicrobial peptides are also produced in recognition of pathogen-associated molecular patterns by Toll receptors in insects and Toll-like receptors in mammals (33). Insects are a tractable surrogate host that may be used to reduce the number of mammals used (33,34), and in particular, Madagascar hissing cockroaches (HCs) have been used in B. mallei, B. pseudomallei, and B. thailandensis virulence studies (35). MNGCs are formed in the hemolymph of HCs upon Burkholderia infection, and the virulence in HCs correlates with that in rodent models of infection (35).…”

“…Insects are a tractable surrogate host that may be used to reduce the number of mammals used (33,34), and in particular, Madagascar hissing cockroaches (HCs) have been used in B. mallei, B. pseudomallei, and B. thailandensis virulence studies (35). MNGCs are formed in the hemolymph of HCs upon Burkholderia infection, and the virulence in HCs correlates with that in rodent models of infection (35). Here, in 4 separate experiments, groups of 10 or 12 HCs were infected with B. thailandensis, left untreated or treated with CLQ, and followed for 7 days.…”

Type VI Secretion Effectors: Methodologies and Biology

“…In B. pseudomallei , transposon insertion mutant library screening revealed the T3SS as the main system related to host virulence, whereas T6SS-1 is important for the virulence in the Madagascar hissing cockroach model, and T6SS-5 may be associated with fitness in mice (Fisher et al, 2012; Gutierrez et al, 2015). Library screening approaches also identified T6SS associated with other phenotypes.…”

Biofilm and motility in response to environmental and host-related signals in Gram negative opportunistic pathogens

“…These data suggest that some virulence factors promoting adherence to eukaryotic cells might have evolved in the original soil habitat of this bacterium, possibly as a mechanism to invade the original hosts for B. pseudomallei , such as amoebae or invertebrates, which, incidentally, are also successfully infected by B. thailandensis (Fisher et al . ). On the other hand, Kamjumphol et al .…”