The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility (CI), which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research1 and relevance to symbiont-induced speciation2,3, as well as control of arbovirus vectors4,5,6 and agricultural pests7, the bacterial genes underlying CI remain unknown. Here, we use comparative and transgenic approaches to demonstrate that two differentially transcribed, codiverging genes in the eukaryotic association module of prophage WO8 from Wolbachia strain wMel recapitulate and enhance CI. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in infected males crossed to uninfected females. Lethality associates with embryonic defects that parallel those of wild type CI and is notably rescued by wMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs Wolbachia’s ongoing utility to control dengue and Zika transmission to humans.

Wolbachia are the most widespread maternally-transmitted bacteria in the animal kingdom. Their global spread in arthropods and varied impacts on animal physiology, evolution, and vector control are in part due to parasitic drive systems that enhance the fitness of infected females, the transmitting sex of Wolbachia. Male killing is one common drive mechanism wherein the sons of infected females are selectively killed. Despite decades of research, the gene(s) underlying Wolbachia-induced male killing remain unknown. Here using comparative genomic, transgenic, and cytological approaches in fruit flies, we identify a candidate gene in the eukaryotic association module of Wolbachia prophage WO, termed WO-mediated killing (wmk), which transgenically causes male-specific lethality during early embryogenesis and cytological defects typical of the pathology of male killing. The discovery of wmk establishes new hypotheses for the potential role of phage genes in sex-specific lethality, including the control of arthropod pests and vectors.

Unidirectional cytoplasmic incompatibility (CI) results in a postfertilization incompatibility between Wolbachia-infected males and uninfected females. CI contributes to reproductive isolation between closely related species and is used in worldwide vector control programs to drastically lower arboviral vector population sizes or to replace populations that transmit arboviruses with those resistant to transmission. Despite decades of research on the factors that influence CI, penetrance is often variable under controlled laboratory conditions in various arthropods, suggesting that additional variables influence CI strength. Here, we demonstrate that paternal D. melanogaster grandmother age influences the strength of CI induced by their sons. Older D. melanogaster females have higher Wolbachia densities and produce offspring with higher Wolbachia densities that associate with stronger CI. This work reveals a multigenerational impact of age on CI and expands our understanding of host-Wolbachia interactions and the biology of CI induced by the Wolbachia strain infecting the most widely used arthropod model, D. melanogaster.

Adaptive antibiotic resistance is a newly described phenomenon by which Acinetobacter baumannii induces efflux pump activity in response to host-associated environmental cues that may, in part, account for antibiotic treatment failures against clinically defined susceptible strains. To that end, during adaptation to growth in human serum, the organism induces approximately 22 putative efflux-associated genes and displays efflux-mediated minocycline tolerance at antibiotic concentrations corresponding to patient serum levels. Here, we show that in addition to minocycline, growth in human serum elicits A. baumannii efflux-mediated tolerance to the antibiotics ciprofloxacin, meropenem, tetracycline, and tigecycline. Moreover, using a whole-cell highthroughput screen and secondary assays, we identified novel serum-associated antibiotic efflux inhibitors that potentiated the activities of antibiotics toward serum-grown A. baumannii. Two compounds, Acinetobacter baumannii efflux pump inhibitor 1 ( ABEPI1) [(E)-4-((4-chlorobenzylidene)amino)benezenesulfonamide] and ABEPI2 [N-tert-butyl-2-(1-tert-butyltetrazol-5-yl)sulfanylacetamide], were shown to lead to minocycline accumulation within A. baumannii during serum growth and inhibit the efflux potential of the organism. While both compounds also inhibited the antibiotic efflux properties of the bacterial pathogen Pseudomonas aeruginosa, they did not display significant cytotoxicity toward human cells or mammalian Ca 2؉ channel inhibitory effects, suggesting that ABEPI1 and ABEPI2 represent promising structural scaffolds for the development of new classes of bacterial antibiotic efflux pump inhibitors that can be used to potentiate the activities of current and future antibiotics for the therapeutic intervention of Gram-negative bacterial infections.

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Staphylococcus aureus is a rapidly growing health threat in the U.S., with resistance to several commonly prescribed treatments. A high-throughput screen identified the antihistamine terfenadine to possess, previously unreported, antimicrobial activity against S. aureus and other Gram-positive bacteria. In an effort to repurpose this drug, structure–activity relationship studies yielded 84 terfenadine-based analogues with several modifications providing increased activity versus S. aureus and other bacterial pathogens, including Mycobacterium tuberculosis. Mechanism of action studies revealed these compounds to exert their antibacterial effects, at least in part, through inhibition of the bacterial type II topoisomerases. This scaffold suffers from hERG liabilities which were not remedied through this round of optimization; however, given the overall improvement in activity of the set, terfenadine-based analogues provide a novel structural class of antimicrobial compounds with potential for further characterization as part of the continuing process to meet the current need for new antibiotics.

Endosymbiotic bacteria in the genus Wolbachia remarkably infect nearly half of all arthropod species. They spread in part because of manipulations of host sexual reproduction that enhance the maternal transmission of the bacteria, including male killing (death of infected males) and unidirectional cytoplasmic incompatibility (CI; death of offspring from infected fathers and uninfected mothers). Recent discoveries identified several genes in prophage WO of Wolbachia (wmk, cifA, and cifB) that fully or partially recapitulate male killing or CI when transgenically expressed in Drosophila melanogaster. However, it is not yet fully resolved if other gene candidates contribute to these phenotypes. Here, we transgenically tested 10 additional gene candidates for their involvement in male killing and/or CI. The results show that despite sequence and protein architecture similarities or comparative associations with reproductive parasitism, transgenic expression of the candidates does not recapitulate male killing or CI. Sequence analysis across Wmk and its closest relatives reveals amino acids that may be important to its function. In addition, evidence is presented to propose new hypotheses regarding the relationship between wmk transcript length and its ability to kill a given host, as well as copy number of wmk homologs within a bacterial strain, which may be predictive of host resistance. Together, these analyses continue to build the evidence for identification of wmk, cifA, and cifB as the major genes that have thus far been shown to cause reproductive parasitism in Wolbachia, and the transgenic resources provide a basis for further functional study of phage WO genes. IMPORTANCE Wolbachia are widespread bacterial endosymbionts that manipulate the reproduction of diverse arthropods to spread through a population and can substantially shape host evolution. Recently, reports identified three prophage WO genes (wmk, cifA, and cifB) that transgenically recapitulate many aspects of reproductive manipulation in Drosophila melanogaster. Here, we transgenically tested 10 additional gene candidates for CI and/or male killing in flies. The results yield no evidence for the involvement of these gene candidates in reproductive parasitism, bolstering the evidence for identification of the cif and wmk genes as the major factors involved in their phenotypes. In addition, evidence supports new hypotheses for prediction of male-killing phenotypes or lack thereof based on wmk transcript length and copy number. These experiments inform efforts to understand the full basis of reproductive parasitism for basic and applied purposes and lay the foundation for future work on the function of an interesting group of Wolbachia and phage WO genes.

Wolbachia are the most widespread bacterial endosymbionts in animals. Within arthropods, these maternally-transmitted bacteria can selfishly hijack host reproductive processes to increase the relative fitness of their transmitting females. One such form of reproductive parasitism called male killing, or the selective killing of infected males, is recapitulated to degrees by transgenic expression of the WO-mediated killing (wmk) gene. Here, we characterize the genotype-phenotype landscape of wmk-induced male killing in D. melanogaster using transgenic expression. While phylogenetically distant wmk homologs induce no sex-ratio bias, closely-related homologs exhibit complex phenotypes spanning no death, male death, or death of all hosts. We demonstrate that alternative start codons, synonymous codons, and notably a single synonymous nucleotide in wmk can ablate killing. These findings reveal previously unrecognized features of transgenic wmk-induced killing and establish new hypotheses for the impacts of post-transcriptional processes in male killing variation. We conclude that synonymous sequence changes are not necessarily silent in nested endosymbiotic interactions with life-or-death consequences.