High-Throughput, Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

Abstract

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a highthroughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD 50 ) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD 50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD 50 . Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD 50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD 50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the ⌬lpp single or the ⌬lpp ⌬msbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD 50 . The mice infected with the ⌬lpp ⌬msbB ⌬rbsA triple mutant at 50 LD 50 were 90% protected upon subsequent challenge with 12 LD 50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s). Yersinia pestis is a tier 1 select agent that leads to three pathodynamic manifestations in humans, namely, bubonic, septicemic, and pneumonic plague (1). Although the disease is endemic in certain regions of the globe (2), the potential use of this organism as a biological warfare agent is a significant worldwide concern. In particular, aerosolized droplets charged with Y. pestis can lead to primary pneumonic plague and subsequent person-toperson spread, with a narrow window for antibiotic intervention (3-5). Consequently, an ideal strategy to combat the disease is to have a vaccine offering long-lasting immunity.Until 1999, a heat-killed plague vaccine composed of Y. pestis 195/P strain was availab...

New Insights into Autoinducer-2 Signaling as a Virulence Regulator in a Mouse Model of Pneumonic Plague

“…The initial finding we reported, that the deletion of the rbsA gene synergistically attenuated Y. pestis CO92 in association with deletions of lpp and msbB genes in a mouse model of pneumonic plague, led us to investigate mechanisms of attenuation beyond the impairment of ribose transport and utilization (11). Since orthologs of the Rbs operon are also associated with AI-2 transport, we examined the effect of combinatorial deletion of lpp , msbB , and rbsA on the levels of AI-2 in the culture supernatants of mutants versus the level in the supernatant of WT CO92.…”

“…Generally, the AI-2 signaling is characterized in a given organism by deleting the gene encoding the primary synthetic enzyme for the AI-2 substrate, LuxS, and observing changes in bacterial virulence phenotypes (10). During the course of our investigation into novel virulence factors of Yersinia pestis , the causative agent of plague, we reported a dramatic increase in attenuation of the Δ lpp Δ msbB Δ rbsA combinatorial deletion mutant in a stringent pneumonic plague mouse model (11). Our earlier studies showed that deletions of lpp , the gene encoding Braun lipoprotein (Lpp), and msbB , a gene encoding a lipopolysaccharide (LPS)-modifying acyltransferase (MsbB), attenuated a highly virulent Y. pestis strain, CO92 (12–14).…”

“…While Lpp activates Toll-like receptor 2 (TLR-2) signaling, MsbB adds lauric acid to the lipid A moiety of LPS to modulate TLR-4 signaling (12). The additional deletion of rbsA (identified during our genome-wide, transposon-based, signature-tagged mutagenesis of Y. pestis CO92 [11]), encoding the ATP binding protein ribose ATP binding cassette (ABC) transporter, led to a further attenuation of the Δ lpp ΔmsbB mutant that was in excess of 10-fold (11). Investigation into the mechanism of the attenuation due to the deletion of rbsA within the rbsBAC operon showed that RbsA was necessary for efficient bacterial growth in a minimal medium limited to a ribose carbon source (11).…”

“…The additional deletion of rbsA (identified during our genome-wide, transposon-based, signature-tagged mutagenesis of Y. pestis CO92 [11]), encoding the ATP binding protein ribose ATP binding cassette (ABC) transporter, led to a further attenuation of the Δ lpp ΔmsbB mutant that was in excess of 10-fold (11). Investigation into the mechanism of the attenuation due to the deletion of rbsA within the rbsBAC operon showed that RbsA was necessary for efficient bacterial growth in a minimal medium limited to a ribose carbon source (11). While RbsA has ATPase activity, its coupling with RbsC, a bacterial membrane-associated protein, actively transports ribose that has been shuttled through the periplasm of the organism by high-affinity association with RbsB (15, 16).…”

Identification of host factors required for Yersinia pestis macrophage intracellular survival and their impact on vacuole maturation, acidification and trafficking.

“…Efforts to identify Y. pestis proteins responsible for intracellular survival by targeting the bacterium itself using mutagenic screens have had limited success, and have only identified the PhoP/Q regulon as required [86,[291][292][293]. We have identified nine Rab GTPases that appear to …”