Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies

Abstract

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host–pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

Comparison of high throughput RNA sequences between Babesia bigemina and Babesia bovis revealed consistent differential gene expression that is required for the Babesia life cycle in the vertebrate and invertebrate hosts

“…In P. falciparum , ROM4 function is related to cleavage of the adhesins of the cell surface during the invasion of RBC ( O’Donnell et al., 2006 ). Babesia bigemina and B. bovis have three to five ROM4, and one ROM6, ROM7 and ROM8 ( Gallenti et al., 2021 ; Gallenti et al., 2022 ). In B. bigemina RNA-seq, kinetes and blood-stages expressed genes encoding ROM proteases ( Table 8 ).…”

Comparative Degradome Analysis of the Bovine Piroplasmid Pathogens Babesia bovis and Theileria annulata

“…Specifically, in B. bovis, the rom4 locus experienced a large expansion, giving rise to a gene family of five paralogous members. Thus, an increased number of serine proteinases of ROM4 can be observed between B. bovis , encoding five ROM4 vs. T. annulata , encoding two ROM4 paralogs [ 67 , 69 ]. While ROM6, located in the mitochondrion, is ubiquitous in eukaryotic organisms, ROM4, 7, and 8 are exclusively present in Aconoidasida.…”