Despite the medical importance of melioidosis, studying B. pseudomallei is still cumbersome, especially in Western countries, where the awareness of melioidosis as a potential bioweapon has led to implementation of stringent security and containment requirements. Nowhere is this more evident than in the United States. The strict regulations governing the acquisition, possession, and use of B. pseudomallei in the United States hinder even basic studies of the physiology, genetics, antimicrobial resistance, etc., of this important yet understudied pathogen and make sharing of mutants difficult because of the permit requirements. Therefore, there is a growing need for safe, attenuated mutants of B. pseudomallei that can be utilized under BSL-2 containment conditions.
As mentioned above, several B. pseudomallei
mutants that exhibit various degrees of attenuation in cell culture or animal models have been identified. These mutants were created mostly for live vaccine studies. Attenuated mutants fall into roughly two categories, mutants that do not express virulence factors (e.g., mutants that do not express capsule [3
], mutants that lack a type IV pilin [15
], and mutants that lack components of the type III secretion system [36
]) and mutants that have metabolic defects (e.g., mutants that have defects in branched-chain amino acid biosynthesis [4
], aromatic compound synthesis [35
], and purine biosynthesis [5
]). Most of these mutants, however, either were not created using select-agent-compliant methods or were not well characterized at the molecular level or thoroughly evaluated in various animal models so that they met the criteria required for an organism to be considered for exclusion from the select-agent list.
In this study we created attenuated mutants, characterized them at the molecular level, and thoroughly evaluated them in various animal models with the goal of defining a strain that meets the criteria for exclusion from the select-agent list. We used select-agent-compliant methods to isolate and characterize defined ΔpurM
mutants. This target was chosen mainly for two reasons. First, transposon-induced B. pseudomallei purM
mutants were shown to be severely replication deficient in cell culture and attenuated in a murine melioidosis model (5
). Second, the purine biosynthetic pathway is a validated target for attenuated mutant construction in bacteria. It has been successfully targeted in attempts to isolate attenuated mutants of Francisella tularensis
), Brucella abortus
), Shigella flexneri
), Actinobacillus pleuropneumoniae
), and Mycobacterium tuberculosis
). In this study we constructed Bp82 and Bp190, which are ΔpurM
derivatives of strains 1026b and K96243, respectively. 1026b and K9623 were originally isolated from human melioidosis patients in Thailand. While 1026b is amenable to genetic manipulation and has been widely used for research in North America, K96243 is the sequenced prototype strain.
In vitro growth experiments demonstrated that Bp82 was an obligate auxotroph and required both adenine and thiamine for normal growth in M9-glucose minimal medium. In contrast, the growth of Bp190 was only partially attenuated in the same medium, and normal growth required addition of both adenine and thiamine. At present, we do not understand why Bp190 is not an obligate adenine auxotroph. One possible explanation is that in contrast to the truncated 99-amino-acid PurM protein present in Bp82, Bp190 PurM has an internal in-frame 38-amino-acid deletion. The 313-amino-acid PurM protein may have enough enzymatic activity to sustain partial growth in minimal media in the absence of adenine and thiamine. We are currently constructing a K96243 ΔpurM strain with the Bp82 allele to test this hypothesis. We did not observe any suppressors of adenine and thiamine auxotrophy in vitro for either Bp82 or Bp190.
Both ΔpurM mutants constructed in this study have an internal deletion which is unlikely to be repaired by natural means. For increased safety and a decreased likelihood of suppression of the ΔpurM mutation, we are currently constructing derivatives of Bp82 and Bp190 that have at least one additional auxotrophic mutation. The growth rates of 1026b and K96243 and their ΔpurM derivatives Bp82 and Bp190 in rich medium were indistinguishable, indicating that the fitness of these strains under these conditions is very similar. These data indicate that ΔpurM mutants are valid surrogates for many basic biological and applied studies (e.g., deciphering antibiotic resistance mechanisms or drug discovery research).
When i.n. inoculation was used, the ΔpurM mutants were avirulent in acute BALB/c infection models even at high challenge doses (up to 106 CFU).
For demonstration of complete safety and lack of virulence in vivo
, it is often necessary to conduct challenge studies with strains of animals that are extremely susceptible to bacterial infection and with immunodeficient animals. The results of the present study show that B. pseudomallei
mutants Bp82 and Bp190 were fully attenuated in hypersusceptible 129/SvEv mice, and Bp82 was also avirulent in the Syrian hamster model. In addition, the mutant strains did not efficiently replicate in vivo
or disseminate following i.n. challenge with high doses. It should also be noted that the animals used in this study were infected via the inhalational challenge route, which is the most lethal route of infection and the route by which healthy laboratory workers would most likely be infected (31
). Moreover, B. pseudomallei
strain Bp82 did not cause mortality in immunodeficient mice, including IFN-γ−/−
mice and SCID mice. Thus, based on very stringent animal challenge criteria, the B. pseudomallei
strains created here are fully attenuated. The attenuation was due solely to the purM
defect since repair of the Bp82 and Bp190 ΔpurM
allele with wild-type sequences resulted in adenine and thiamine prototrophy and restored virulence.
In summary, extensive in vitro
characterization and stringent animal challenge experiments showed that both B. pseudomallei
derivatives constructed and tested in this study are, in principle, viable candidates for exclusion from select-agent lists. However, given the overall evidence, we consider the B. pseudomallei
mutant Bp82 the superior attenuated strain candidate. This mutant is fully attenuated in vitro
when it is grown in adenine- and thiamine-deficient growth medium. In addition, it is avirulent in vivo
, even following high-dose challenge of extremely susceptible wild-type and immunodeficient animals. Moreover, this mutant does not replicate in vivo
and also does not establish chronic infections. Thus, we concluded that to date, B. pseudomallei
mutant Bp82 is the most viable candidate strain for exclusion from select-agent lists and with good laboratory practice is safe for use under BSL-2 conditions. Federal regulations permit such exclusions from the list of select biological agents in cases when it has been established that an attenuated strain of a select biological agent does not pose a severe threat to public health and safety, animal health, or animal products. Unlike the situation for B. mallei
, for which there are variants that are severely attenuated for virulence in their natural host and thus are likely candidates for exclusion from the select-agent list (32
), clinically attenuated B. pseudomallei
strains that grow normally in laboratory media have yet to be discovered. Until such strains are discovered, genetically engineered and well-characterized strains such as the strain described here are the only candidates for consideration for exclusion and provide useful tools for the extended research community.