An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands

Abstract

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference-mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum-infected mice. A. phagocytophilum migrated normally from A. phagocytophilum-infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

Figure 1.

Anaplasma phagocytophilum selectively alters salp gene expression. (a) The expression levels of 14 salp genes in the salivary glands of A. phagocytophilum–infected (I) Ixodes scapularis nymphs were compared with uninfected (UI) nymphs at 24 and 72 h after feeding using RT-PCR. (b) Verification of the up-regulation of salp16 expression in A. phagocytophilum–infected single tick salivary glands at 24 h after infection by RT-PCR. (c) Expression of salp16 in the salivary glands of uninfected and infected unfed ticks by quantitative RT-PCR. (d) Expression of salp16 at 24 and 72 h in the salivary glands of uninfected and infected ticks fed on naive mice or uninfected ticks fed on infected mice by quantitative RT-PCR. Results are expressed as the means ± SEM (error bars) from three independent experiments. P < 0.05 was considered statistically significant (Student's t test).

Figure 2.

Silencing of the salp16 gene using RNAi reduces the acquisition of Anaplasma phagocytophilum by the tick. The efficiency and specificity of RNAi-dependent knockdown of the salp16 gene was assessed using RT-PCR (a) and immunoblotting (b). (a) The expression of salp25D, salp14, salp20, salp25B, salp26A, and salp9 in salp16-deficient tick salivary glands. As shown in b, the specificity of knockdown was confirmed using a duplicate immunoblot probed with Salp14 antisera. (c and d) RT-PCR– (c) and quantitative RT-PCR (d)–based demonstrations of the reduced ability of salp16-deficient ticks to acquire A. phagocytophilum from infected mice. RNAi-injected ticks were fed on infected mice for 72 h, and the level of A. phagocytophilum was assessed in the salivary gland using RT-PCR with the P44 gene as the marker. (e) RT-PCR showing the ablation of salp25D after the injection of ticks with a salp25D RNAi construct. (f) The acquisition of A. phagocytophilum by salp25D-depleted ticks. Results are expressed as the mean ± SEM (error bars) from three independent experiments. P < 0.05 was considered statistically significant (Student's t test). M, mock; KO, knockout.

Figure 3.

Silencing of salp16 expression does not affect the acquisition of B. burgdorferi. salp16 RNAi–injected ticks were allowed to feed on B. burgdorferi–infected mice, and the guts were analyzed at 72 h after infection. (a) RT-PCR–based confirmation of the silencing of salp16 in the knockout tick guts. (b) Analysis of the levels of B. burgdorferi in the gut of knockout ticks (KO) in comparison with mock-injected ticks (M) by quantitative RT-PCR analysis. flaB gene expression was used to measure the levels of B. burgdorferi. Results are means ± SEM (error bars) from one representative experiment. (c) Confirmation of the levels of B. burgdorferi in the gut of mock and knockout ticks by confocal microscopy. Gut samples from mock and knockout ticks were probed with FITC-labeled anti–B. burgdorferi antibody (green), and nuclei were stained with TO-PRO. (d) Knockout of salp16 in the salivary glands and (f) gut of ticks fed on coinfected mice for 72 h assessed by RT-PCR. The levels of A. phagocytophilum (e) and B. burgdorferi (g) in ticks fed on coinfected mice was performed by measuring the levels of A. phagocytophilum P44 and B. burgdorferi flaB. Results are the means ± SEM from three quantitative PCR experiments. P < 0.05 was considered statistically significant (Student's t test).

Figure 4.

A. phagocytophilum does not use Salp16 in the mammalian host before acquisition by the arthropod. (a) Binding of Salp16 with human neutrophils. Extracts of human neutrophils or BSA (control) were incubated with recombinant Salp16, and a binding study was performed as described in Materials and methods. Bars represent the mean ± SEM (error bars) from three studies. (b) A neutrophil chemotaxis assay using Salp16. Freshly isolated human neutrophils were used as chemoattractants in 96-well Neuro Probe TX chambers together with recombinant IL-8 as a positive control and medium as a negative control as described in Materials and methods. The results are expressed as the migration index after subtraction of background values (medium alone). The means ± SEM of three independent studies are shown. (c) Immunoblot showing that recombinant Salp16 (expressed in bacteria) is recognized by tick immune sera. Lane 1 shows recombinant Salp16 (GST fusion) probed with rabbit tick immune sera; lane 2 shows GST protein probed with tick immune sera; and lane 3 shows recombinant Salp16 probed with Salp16 antisera. (d) Comparison of the number of neutrophils entering the mock and salp16 knockout (KO) ticks. After the feeding of mock and salp16 knockout ticks on infected mice, the number of neutrophils within the whole ticks was assessed by measuring the lactoferrin (neutrophil-specific gene) levels (normalized to mouse β-actin gene and tick β-actin gene) by quantitative RT-PCR analysis. The results are expressed as means ± SEM from three independent experiments.

Figure 5.

A. phagocytophilum requires Salp16 for the initial infection of the tick salivary gland. A time course assessment of the levels of A. phagocytophilum within the salivary glands and guts of salp16-deficient (KO) and mock (M) ticks fed on infected mice for 24, 48, and 72 h is shown. Repression of salp16 in the salivary glands of ticks fed for 24 (a), 48 (c), and 72 h (e) by RT-PCR. The levels of A. phagocytophilum P44 within the salivary glands of ticks fed for 24 (b), 48 (d), and 72 h (f) by quantitative RT-PCR. Repression of salp16 within the corresponding gut samples of ticks fed for 24 (g), 48 (i), and 72 h (k) by RT-PCR. The levels of A. phagocytophilum P44 in the gut samples of ticks fed for 24 (h), 48 (j), and 72 h (l). (m) Levels of A. phagocytophilum P44 in the salivary glands of ticks on days 5, 7, and 9 after feeding, and (n) levels of salp16 in the salivary glands at the respective time points. The results are expressed as means ± SEM (error bars) from a representative experiment. P < 0.05 was considered statistically significant (Student's t test).

Figure 6.

salp16 is not required for the maintenance of A. phagocytophilum within infected tick salivary glands. salp16 RNAi was injected into A. phagocytophilum–infected ticks and were analyzed either in an unfed state or in a 72-h fed state. (a) Knockdown of salp16 expression in A. phagocytophilum–infected unfed ticks by RT-PCR, and (b) quantification of A. phagocytophilum P44 levels by quantitative RT-PCR analysis. (c) Knockdown of salp16 expression in 72-h fed ticks by RT-PCR, and (d) quantitative RT-PCR data showing A. phagocytophilum P44 levels. Results are means ± SEM (error bars) from three independent experiments. P < 0.05 was considered statistically significant (Student's t test). M, mock; KO, knockout.

Figure 7.

Confocal microscopy showing the distribution of Salp16 within engorged tick salivary glands. Ticks were fed for 72 h, and salivary glands were isolated and probed with anti-GST antisera (negative control; a) and anti-Salp16 polyclonal antisera (b). Binding was visualized using tetramethylrhodamine isothiocyanate–conjugated secondary antibody, and the samples were counterstained with nuclear stain DAPI.