An NF-κB-based high-throughput screen identifies piericidins as inhibitors of the Yersinia pseudotuberculosis type III secretion system.

The type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibited Yersinia pseudotuberculosis from triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As the Yersinia T3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of the Y. pseudotuberculosis T3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growth in vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics in Yersinia. In addition, when Yersinia was incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS.

Chemical inhibitors of the type three secretion system: disarming bacterial pathogens.

The recent and dramatic rise of antibiotic resistance among bacterial pathogens underlies the fear that standard treatments for infectious disease will soon be largely ineffective. Resistance has evolved against nearly every clinically used antibiotic, and in the near future, we may be hard-pressed to treat bacterial infections previously conquered by "magic bullet" drugs. While traditional antibiotics kill or slow bacterial growth, an important emerging strategy to combat pathogens seeks to block the ability of bacteria to harm the host by inhibiting bacterial virulence factors. One such virulence factor, the type three secretion system (T3SS), is found in over two dozen Gram-negative pathogens and functions by injecting effector proteins directly into the cytosol of host cells. Without T3SSs, many pathogenic bacteria are unable to cause disease, making the T3SS an attractive target for novel antimicrobial drugs. Interdisciplinary efforts between chemists and microbiologists have yielded several T3SS inhibitors, including the relatively well-studied salicylidene acylhydrazides. This review highlights the discovery and characterization of T3SS inhibitors in the primary literature over the past 10 years and discusses the future of these drugs as both research tools and a new class of therapeutic agents.

High-Throughput Analysis of Gene Function in the Bacterial Predator Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a bacterial predator capable of killing and replicating inside most Gram-negative bacteria, including antibiotic-resistant pathogens. Despite growing interest in this organism as a potential therapeutic, many of its genes remain uncharacterized. Here, we perform a high-throughput genetic screen with B. bacteriovorus using transposon sequencing (Tn-seq) to explore the genetic requirements of predation. Two hundred one genes were deemed essential for growth in the absence of prey, whereas over 100 genes were found to be specifically required for predative growth on the human pathogens Vibrio cholerae and Escherichia coli in both planktonic and biofilm states. To further this work, we created an ordered-knockout library in B. bacteriovorus and developed new high-throughput techniques to characterize the mutants by their stage of deficiency in the predator life cycle. Using microscopy and flow cytometry, we confirmed 10 mutants defective in prey attachment and eight mutants defective in prey rounding. The majority of these genes are hypothetical and previously uncharacterized. Finally, we propose new nomenclature to group B. bacteriovorus mutants into classes based on their stage of predation defect. These results contribute to our basic understanding of bacterial predation and may be useful for harnessing B. bacteriovorus to kill harmful pathogens in the clinical setting.IMPORTANCEBdellovibrio bacteriovorus is a predatory bacterium that can kill a wide range of Gram-negative bacteria, including many human pathogens. Given the global rise of antibiotic resistance and dearth of new antibiotics discovered in the past 30 years, this predator has potential as an alternative to traditional antibiotics. For many years, B. bacteriovorus research was hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established. Here, we comprehensively identify and characterize predator genes required for killing bacterial prey, as well as genes that interfere in this process, which may allow us to design better therapeutic predators. Based on our study, we and other researchers may ultimately be able to genetically engineer strains that have improved killing rates, target specific species of prey, or preferentially target prey in the planktonic or biofilm state.

Vibrio cholerae motility exerts drag force to impede attack by the bacterial predator Bdellovibrio bacteriovorus.

The bacterial predator Bdellovibrio bacteriovorus is evolved to attack and kill other bacteria, including the human intestinal pathogen Vibrio cholerae. Although B. bacteriovorus exhibit a broad prey range, little is known about the genetic determinants of prey resistance and sensitivity. Here we perform a genetic screen on V. cholerae and identify five pathways contributing to predation susceptibility. We find that the essential virulence regulators ToxR/S increase susceptibility to predation, as mutants of these genes are more resistant to predation. We observe by flow cytometry that lipopolysaccharide is a critical defense, as mutants lacking O-antigen are rapidly attacked by predatory B. bacteriovorus. Using polymer solutions to alter media viscosity, we find that when B. bacteriovorus attacks motile V. cholerae, increased drag forces slow its ability to prey. These results provide insights into key prey resistance mechanisms, and may be useful in the application of B. bacteriovorus in treating infections.

Bacterial internalization is required to trigger NIK-dependent NF-κB activation in response to the bacterial type three secretion system.

Infection of human cells with Yersinia pseudotuberculosis expressing a functional type III secretion system (T3SS) leads to activation of host NF-κB. We show that the Yersinia T3SS activates distinct NF-κB pathways dependent upon bacterial subcellular localization. We found that wildtype Yersinia able to remain extracellular triggered NF-κB activation independently of the non-canonical NF-κB kinase NIK in HEK293T cells. In contrast, Yersinia lacking the actin-targeting effectors YopEHO, which become internalized into host cells, induce a NIK-dependent response and nuclear entry of the non-canonical NF-κB subunit p52. Blocking actin polymerization and uptake of effector mutant bacteria using cytochalasin D shifted the host NF-κB response from NIK-independent to primarily NIK-dependent. We observed similar results using Pseudomonas aeruginosa, which expresses a related T3SS and the actin-targeting effector ExoT. As the NF-κB response of HEK293T cells to effectorless Yersinia has been used both as a screening tool for chemical inhibitors of the T3SS and for bacterial forward genetic screens, a better understanding of this response is important for tool optimization and interpretation.

Piericidin A1 Blocks Yersinia Ysc Type III Secretion System Needle Assembly.

The type III secretion system (T3SS) is a bacterial virulence factor expressed by dozens of Gram-negative pathogens but largely absent from commensals. The T3SS is an attractive target for antimicrobial agents that may disarm pathogenic bacteria while leaving commensal populations intact. We previously identified piericidin A1 as an inhibitor of the Ysc T3SS in Yersinia pseudotuberculosis. Piericidins were first discovered as inhibitors of complex I of the electron transport chain in mitochondria and some bacteria. However, we found that piericidin A1 did not alter Yersinia membrane potential or inhibit flagellar motility powered by the proton motive force, indicating that the piericidin mode of action against Yersinia type III secretion is independent of complex I. Instead, piericidin A1 reduced the number of T3SS needle complexes visible by fluorescence microscopy at the bacterial surface, preventing T3SS translocator and effector protein secretion. Furthermore, piericidin A1 decreased the abundance of higher-order YscF needle subunit complexes, suggesting that piericidin A1 blocks YscF needle assembly. While expression of T3SS components in Yersinia are positively regulated by active type III secretion, the block in secretion by piericidin A1 was not accompanied by a decrease in T3SS gene expression, indicating that piericidin A1 may target a T3SS regulatory circuit. However, piericidin A1 still inhibited effector protein secretion in the absence of the T3SS regulator YopK, YopD, or YopN. Surprisingly, while piericidin A1 also inhibited the Y. enterocolitica Ysc T3SS, it did not inhibit the SPI-1 family Ysa T3SS in Y. enterocolitica or the Ysc family T3SS in Pseudomonas aeruginosa. Together, these data indicate that piericidin A1 specifically inhibits Yersinia Ysc T3SS needle assembly. IMPORTANCE The bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood. Deciphering how some natural products, such as the microbial metabolite piericidin, inhibit type III secretion can provide important insight into how the T3SS functions or is regulated. Taking this approach, we investigated the ability of piericidin to block T3SS function in several human pathogens. Surprisingly, piericidin selectively inhibited the Ysc family T3SS in enteropathogenic Yersinia but did not affect the function of a different T3SS within the same species. Furthermore, piericidin specifically blocked the formation of T3SS needles on the bacterial surface without altering the localization of several other T3SS components or regulation of T3SS gene expression. These data show that piericidin targets a mechanism important for needle assembly that is unique to the Yersinia Ysc T3SS.