Targeted mutagenesis in Anaplasma marginale to define virulence and vaccine development against bovine anaplasmosis.

Tick-borne Anaplasma species are obligate, intracellular, bacterial pathogens that cause important diseases globally in people, agricultural animals, and dogs. Targeted mutagenesis methods are yet to be developed to define genes essential for these pathogens. In addition, vaccines conferring protection against diseases caused by Anaplasma species are not available. Here, we describe a targeted mutagenesis method for deletion of the phage head-to-tail connector protein (phtcp) gene in Anaplasma marginale. The mutant did not cause disease and exhibited attenuated growth in its natural host (cattle). We then assessed its ability to confer protection against wild-type A. marginale infection challenge. Additionally, we compared vaccine protection with the mutant to that of whole cell A. marginale inactivated antigens as a vaccine (WCAV) candidate. Upon infection challenge, non-vaccinated control cattle developed severe disease, with an average 57% drop in packed cell volume (PCV) between days 26-31 post infection, an 11% peak in erythrocytic infection, and apparent anisocytosis. Conversely, following challenge, all animals receiving the live mutant did not develop clinical signs or anemia, or erythrocyte infection. In contrast, the WCAV vaccinees developed similar disease as the non-vaccinees following A. marginale infection, though the peak erythrocyte infection reduced to 6% and the PCV dropped 43%. This is the first study describing targeted mutagenesis and its application in determining in vivo virulence and vaccine development for an Anaplasma species pathogen. This study will pave the way for similar research in related Anaplasma pathogens impacting multiple hosts.

Biochemical characterization of ClpB and DnaK from Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an intracellular tick-transmitted bacterial pathogen that infects neutrophils in mammals and causes granulocytic anaplasmosis. In this study, we investigated the molecular chaperones ClpB and DnaK from A. phagocytophilum. In Escherichia coli, ClpB cooperates with DnaK and its co-chaperones DnaJ and GrpE in ATP-dependent reactivation of aggregated proteins. Since ClpB is not produced in metazoans, it is a promising target for developing antimicrobial therapies, which generates interest in studies on that chaperone's role in pathogenic bacteria. We found that ClpB and DnaK are transcriptionally upregulated in A. phagocytophilum 3-5 days after infection of human HL-60 and tick ISE6 cells, which suggests an essential role of the chaperones in supporting the pathogen's intracellular life cycle. Multiple sequence alignments show that A. phagocytophilum ClpB and DnaK contain all structural domains that were identified in their previously studied orthologs from other bacteria. Both A. phagocytophilum ClpB and DnaK display ATPase activity, which is consistent with their participation in the ATP-dependent protein disaggregation system. However, despite a significant sequence similarity between the chaperones from A. phagocytophilum and those from E. coli, the former were not as effective as their E. coli orthologs during reactivation of aggregated proteins in vitro and in supporting the survival of E. coli cells under heat stress. We conclude that the A. phagocytophilum chaperones might have evolved with distinct biochemical properties to maintain the integrity of pathogenic proteins under unique stress conditions of an intracellular environment of host cells.

Genetically modified live vaccine offers protective immunity against wild-type Anaplasma marginale tick-transmission challenge.

Anaplasma marginale is a tick-borne pathogen of cattle that causes bovine anaplasmosis in tropical and subtropical regions throughout the world. Killed vaccines derived from infected erythrocytes have been used for control of this disease with limited success. Recently, we described a targeted deletion mutation in the phage head-to-tail connector protein gene of A. marginale which caused bacterial attenuation in vivo and provided protection as a modified live vaccine (MLAV). Following intravenous injection of susceptible steers, the MLAV induced protective immunity against disease progression. In the current study, we demonstrated that the immunity resulting from MLAV in cattle prevents the disease progression resulting from virulent A. marginale intrastadial transmission from infected Dermacentor variabilis male ticks. The nonimmunized control steers receiving the infection from ticks developed fever, lethargy, and inappetence for several days post tick exposure with significant decreases in the packed cell volume and increases in bacteremia. In contrast, the MLAV immunized steers remained healthy after being challenged with infected ticks and this group of animals had a significant reduction in bacteremia as compared with the controls. This study demonstrated that the A. marginale MLAV provided protection against acute tick-transmitted anaplasmosis, in addition to protection documented in steers challenge-exposed with infected blood as reported previously.