The
resuscitation-promoting factor (Rpf), a secretory
protein first reported in Micrococcus luteus, plays a critical role in mycobacterial survival and
infection. There are five functionally redundant Rpf-like
proteins identified in M.
tuberculosis (Mtb). All these Rpfs share a conserved Rpf domain (Rpfd) composed of approximately 70
amino acids, which possesses the same
biological functions as the full-length Rpf
protein.
Glutamic acid at position 54 in Rpfd (E54) has been implicated in mediating multiple physiological processes, and a single amino acid substitution at residue E54 can affect the
protein biological activity. In order to determine the effects of different amino acid substitutions of E54 in Rpfd on its immunogenic activity, we generated three recombinant Rpfd mutants, Rpfd1 (E54K), Rpfd2 (E54A) and Rpfd3 (E54K and D48A), based on
T-cell epitope prediction and tested their potential protective/
therapeutic effects against Mtb in mice. Our results demonstrated that replacement of E54 by different
amino acids in Rpfd distinctively influenced its
resuscitation-promoting activities and Th1-type immune responses induced in mice. Administration of Rpfd2 mutant enhanced Th1-type cellular responses (IFN-γ and IL-2) in mice (P < 0.05, Rpfd2 versus control) and provided effective protection against Mtb in mice by significantly inhibiting the growth of Mtb during the initial stage of
infection. Four weeks after the challenge, the slightest pathological injury in lung was observed in the Rpfd2-immunized group among all three Rfpd mutant-immunized groups. Furthermore, Rpfd2
therapy significantly decreased the bacterial load in lung and alleviated histopathological damage in Mtb-infected mice. Together, our results suggest Rpfd2 as a novel effective
vaccine candidate against Mtb.