Disruption of VirB6 Paralogs in Anaplasma phagocytophilum Attenuates Its Growth

Abstract

Many pathogenic bacteria translocate virulence factors into their eukaryotic hosts by means of Type 4 Secretion Systems (T4SS) spanning the inner and outer membranes. Genes encoding components of these systems have been identified within the Order Rickettsiales based upon their sequence similarities to other prototypical systems. Anaplasma phagocytophilum are obligate intracellular, tick-borne bacteria that are members of this Order. The organization of these components at the genomic level was determined in several Anaplasma phagocytophilum strains, showing overall conservation, with the exceptions of virB2 and virB6 genes. The virB6 loci are characterized by the presence of four virB6 copies (virB6-1 through virB6-4) arranged in tandem within a gene cluster known as the sodB-virBs operon. Interestingly, the virB6-4 gene varies significantly in length among different strains due to extensive tandem repeats at the 3′ end. To gain understanding of how these enigmatic virB6 genes function in A. phagocytophilum we investigated their expression in infected human and tick cells. Our results show that these genes are expressed by A. phagocytophilum replicating in both cell types, and that VirB6-3 and VirB6-4 proteins are surface-exposed. Analysis of an A. phagocytophilum mutant carrying the Himar1 transposon within the virB6-4 gene, demonstrated that the insertion not only disrupted its expression but also exerted a polar effect in the sodB-virBs operon. Moreover, the altered expression of genes within this operon was associated with the attenuated in vitro growth of A. phagocytophilum in human and tick cells, indicating the importance of these genes in the physiology of this obligate intracellular bacterium in such different environments. IMPORTANCE Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens. The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4322 (GenBank YYU_01815) and 9935 (P029_01630) amino acids. To understand how the T4SS is used in A. phagocytophilum we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells is supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library.

Transposon mutagenesis of Rickettsia felis sca1 confers a distinct phenotype during flea infection

“…As it is known that transposon insertions can have polar effects on adjacent genes [ 32 , 34 , 35 ], primers were designed to amplify cDNA of portions of 3 genes located downstream of sca1 , RF_0023 , RF_0024 , and RF_0025 ( S1A Fig ). Of interest, only transcripts from RF_0023 were detected in R .…”

“…As it is known that transposon insertions can have polar effects on adjacent genes [32,34,35], primers were designed to amplify cDNA of portions of 3 genes located downstream of sca1, RF_0023, RF_0024, and RF_0025 (S1A Fig) . Of interest, only transcripts from RF_0023 were detected in R. felis WT, where the absence of transcripts was observed for R. felis sca1::tn (S1B Fig) . No amplification of rickettsial DNA was detected in no reverse transcriptase controls. Thus, the data suggests the Himar1 insertion has altered sca1 gene transcription and subsequently affected transcription of a neighboring gene.…”

AnAnaplasma phagocytophilumT4SS effector, AteA, is essential for tick infection

“…As a control strain, we used another mutant, which contains the Himar1 transposon in an intergenic location. This strain has been shown to be phenotypically equivalent to wild-type A. phagocytophilum (27, 28). Our analysis confirmed that transcription of ateA was abolished in the ateA ::Himar1 mutant, but transcription of housekeeping genes, rpoB and groEL , and the conserved Anaplasma gene encoding major surface protein 5, msp5 , were all unaffected (Figure 1B).…”

“…We therefore used qRT-PCR with primers targeting non-repetitive sequences to quantify ateA expression patterns from A. phagocytophilum grown in HL60 and ISE6 cells. Housekeeping A. phagocytophilum genes, rpoB and groEL , are equally expressed when grown in either mammalian or tick cell lines (7, 27), and were therefore used as baseline controls for expression. Our findings demonstrate that ateA expression was >8 fold higher during growth in tick cells than in mammalian cells (Figure 1A).…”

“…HL60 and ISE6 host cell gDNA was measured targeting genes tlr9 (toll-like receptor 9) and crt (calreticulin). Respectively (27) (Table S1).…”