Targets for the diagnosis of Acanthamoeba eye infections include four cyst wall proteins and the mannose-binding domain of the trophozoite mannose-binding protein

Abstract

Acanthamoebae, which are free-living amoebae, cause corneal inflammation (keratitis) and blindness, if not quickly diagnosed and effectively treated. The walls of Acanthamoeba cysts contain cellulose and have two layers connected by conical ostioles. Cysts are identified by in vivo confocal microscopy of the eye or calcofluor-white- or Giemsa-labeling of corneal scrapings, both of which demand great expertise. Trophozoites, which use a mannose-binding protein to adhere to keratinocytes, are identified in eye cultures that delay diagnosis and treatment. We recently used structural and experimental methods to characterize cellulose-binding domains of Luke and Leo lectins, which are abundant in the inner layer and ostioles. However, no antibodies have been made to these lectins or to a Jonah lectin and a laccase, which are abundant in the outer layer. Here, confocal microscopy of rabbit antibodies (rAbs) to recombinant Luke, Leo, Jonah, and laccase supported localizations of GFP-tagged proteins in walls of transfected Acanthamoebae. rAbs efficiently detected calcofluor white-labeled cysts of 10 of the 11 Acanthamoeba isolates tested, including six T4 genotypes that cause most cases of keratitis. Further, laccase shed into the medium during encystation was detected by an enzyme-linked immunoassay. Structural and experimental methods identified the mannose-binding domain (ManBD) of the Acanthamoeba mannose-binding protein, while rAbs to the ManBD efficiently detected DAPI-labeled trophozoites from all 11 Acanthamoeba isolates tested. We conclude that antibodies to four cyst wall proteins and the ManBD efficiently identify Acanthamoeba cysts and trophozoites, respectively.IMPORTANCEFree-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of unlabeled cysts by in vivo confocal microscopy of the eye or calcofluor-white (CFW) labeled cysts by fluorescence microscopy of corneal scrapings. Alternatively, Acanthamoeba infections are diagnosed by the identification of trophozoites in eye cultures. Here, we showed that rabbit antibodies (rAbs) to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently identify CFW-labeled cysts of 10 of the 11 Acanthamoeba isolates tested. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rAbs to the mannose-binding domain (ManBD) of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identify DAPI-labeled trophozoites of all 11 Acanthamoeba isolates tested. In summary, four wall proteins and the ManBD appear to be excellent targets for the diagnosis of Acanthamoeba cysts and trophozoites, respectively.

Keywords: Acanthamoeba; AlphaFold; ELISA; cyst; cyst wall proteins; diagnosis; keratitis; lateral gene transfer; mannose-binding protein; rabbit antibodies; trophozoite.

Fig 1

Double labels show rabbit antibodies (rAbs) to Jonah-1, Luke-2, laccase-1, and Leo-A localize to similar locations in intact cysts and broken cyst walls as do tagged proteins expressed under their own promoter in transfected Acanthamoebae. (A) AlphaFold with confidence colored shows the single BHF, which was used to make the EcMBP-Jonah-1 fusion-protein for production of anti-Jonah-1 rAbs. The diagram shows the N-terminal signal peptide (purple) and low complexity Thr-rich domain (orange). (B) AlphaFold shows N- and C-terminal BJRFs (BJRF-N and BJRF-C) connected by a low complexity spacer, which were used to make the EcMBP-Luke-2 fusion-protein to produce anti-Luke-2 rAbs. The diagram shows the N-terminal signal peptide (purple) and the low-complexity Ser-rich domain. (C) The first plasmid for double-labeling cyst walls contains full-length Jonah-1 cDNA with 574 bp of its 5′ UTR and a C-terminal mCherry tag head-to-head with full-length Luke-2 cDNA with 446 bp of its 5′ UTR and a C-terminal GFP tag (33). (D) Double labels show Jonah-1-mCherry is present in the ectocyst layer (yellow arrowheads) of intact and broken cysts, while Luke-GFP predominates in the ostioles (green arrowheads) of both preparations. CFW marks the endocyst layer (red arrowheads). (E) Anti-Jonah-1 rAbs (red) bind to the ectocyst layer of intact and broken cysts, while anti-Luke-2 rAbs (green) bind to ostioles of intact cysts and to the endocyst layer of broken walls. (F) Rabbit pre-bleeds fail to bind to intact cysts and broken walls. (G) AlphaFold shows the first copper oxidase domain (CuRO-1), which was used to make EcMBP-CuRO-1 for production of anti-laccase-1 rAbs. The diagram shows three CuRO domains in laccase-1. (H) AlphaFold shows N- and C-terminal sets of 4DKs (4DK-N and 4DK-C), which were used to make EcMBP-Leo-A for production of anti-Leo-A rAbs. The diagram shows the short spacer between sets of 4DKs of Leo-A. (I) The second plasmid for double-labeling cyst walls contains full-length laccase-1 cDNA with 405 bp of its 5′ UTR and a C-terminal RFP tag head-to-head with full-length Leo-A cDNA with 486 bp of its 5′ UTR and a C-terminal GFP. (J) Laccase-1-RFP is in the ectocyst layer of intact and broken cysts, while Leo-A-GFP is present in the endocyst layer and ostioles. (K) Anti-laccase-1 rAbs (red) bind to the ectocyst layer of intact and broken cysts, while anti-Leo-A rAbs (green) bind to the endocyst layer of intact cysts and to ostioles in broken walls. Scale bars for panels D–F, J, and K are 5 µm.

Fig 2

High-power confocal micrographs of 11 Acanthamoeba isolates show anti-Jonah-1 and anti-laccase-1 rAbs consistently bind to the surface of cysts, while anti-Luke-2 and anti-Leo-A rAbs bind to the endocyst layer and ostioles of some cysts (expected) and to the ectocyst layer of other cysts (unexpected). (A and B) The anti-Jonah-1 rAbs bind in a patchy distribution to the surface of cysts of 11 Acanthamoeba isolates, while the anti-laccase-1 rAbs bind in a homogenous manner. CFW labels the endocyst layer. (C and D) Both the anti-Luke-2 and anti-Leo-A rAbs bind to the ostioles and endocyst layer of some cysts of some genotypes (e.g., T4-Neff, T11, T18, and T2/6C) and the ectocyst layer of other cysts (e.g., T4A-1/2, T4B1/2, T4C, T4D, and T1) (see Fig. S3 and File S1 for characterization of genotypes). Possible reasons for the different patterns of binding of rAbs to Luke-2 and Leo-A are discussed in Results. A single scale bar for panels A–D is 5 µm. Random fields of rAbs binding to 11 Acanthamoeba isolates are shown in Fig. 3, while percentages of CFW-tagged cysts detected are shown in Fig. 4A.

Fig 3

Low-power confocal micrographs show examples of random fields used to determine how efficiently rabbit antibodies to wall proteins detect calcofluor-white-labeled cysts of 10 of the 11 isolates of Acanthamoeba. (A–D) Cysts of 11 Acanthamoeba isolates (shown at high power in Fig. 2) were labeled with protein A-purified antibodies tagged with Alexa Fluor 488, and the percentage of CFW-tagged cysts detected was counted for two experiments and plotted in Fig. 4A. The purpose of these low-power images is to give an overview of the brightness and homogeneity of the binding of the four rAbs to the 11 Acanthamoeba isolates. Note that for counting, individual cysts were visualized at nearly the magnification of cysts in Fig. 2. A single scale bar for panel A–D is 50 µm.

Fig 4

Plots show rAbs to Jonah-1, laccase-1, Luke-2, and Leo-A efficiently detect CFW-labeled cysts, while Western blots and ELISAs identify laccase-1 shed into the medium of encysting Ac. (A) The plot shows the percentage of CFW-labeled cysts detected by four rAbs in three independent experiments (average plus SEM), in which 100+ cysts were counted in random low-power fields (Fig. 3). The rAbs to Luke-2, Leo-A, and laccase-1 each detect >95% of cysts of 10 of the 11 Acanthamoeba isolates evaluated, while the rAbs to Jonah-1 are slightly less efficient in detecting 3 isolates of Acanthamoeba. The exception is genotype T4C, which is poorly detected with all four rAbs, suggesting a problem with its cyst formation. (B) Rabbit antibodies to four wall proteins also efficiently detect Ac cysts using conventional IFA microscopy. Scale bar for high power is 5 µm and for low power is 50 µm. (C) Western blots show pre-bleeds from rabbits (negative control) fail to bind to proteins of trophozoites or cysts, while rAbs to Jonah-1, Luke-2, Leo-A, and laccase-1 fail to bind to trophozoite proteins (a second negative control) but bind well to cyst proteins of the Neff strain. As discussed in detail in the Results, the anti-Jonah-1 rAbs bind to the expected 55 kDa band (red arrowhead) and two lower mol wt bands (blue arrowheads), while the anti-Luke-2 rAbs bind to a heavy and broad 50 kDa band (red arrowhead), which is greater than the expected size of 27 kDa. The anti-Leo-A rAbs bind to a 13 kDa band (red arrowhead), which is slightly smaller than the expected size of 17 kDA, while the anti-laccase-1 rAbs bind to a 75 kDa band (red arrowhead), which is slightly bigger than the expected size of 64 kDa, as well as to a less abundant 55 kDa band (blue arrowhead). (D) Western blots of whole cell lysates with rAbs (left) show laccase-1 and Jonah-1 are made early in encystation, while Luke-1 and Leo-A are made later in encystation. These results are consistent with the expression of tagged proteins (33). Western blots of culture supernatants (right) show that intact laccase-1 is abundant throughout encystation, while a degraded form of Jonah-1 is abundant at 48- and 72-h encystation. In contrast, neither Luke-2 nor Leo-A are present in the culture medium (green arrowheads). (E) Consistent with Western blots, direct ELISAs of culture supernatants detected laccase-1 throughout encystation; Jonah-1 was detected at 48- and 72-h encystation, while neither Luke-2 nor Leo-A were detected. (F) A “leaky and sealed” model of cyst wall formation suggests some of laccase-1 (and Jonah-1), which is made early, remains bound to the partially completed outer layer of the wall, while the remaining laccase-1 is secreted into the medium. In contrast, Luke-2 (and Leo-A), which is made later, either binds to the partially completed inner layer of the wall or is trapped in the completed outer layer.

Fig 5

The antiparallel β-sandwich (ABS) of the Acanthamoeba mannose-binding protein (AcMBP) binds mannose-agarose resin and human corneal limbal epithelial (HCLE) cells and so is the mannose-binding domain (ManBD). (A) AlphaFold with the confidence-colored shows AcMBP contains an N-terminal ABS and CRRs composed of anti-parallel β-strands. (B and C) The ABS has four loops linked by disulfides, while disulfides also link ends of β-strands in a representative segment of the CRRs. (D and E) Foldseek and PyMOL show that the ABS of AcMBP closely matches the same domain in an uncharacterized protein of Myxococcus and is like ManBD of Burholderia, which has been crystallized (PDB 2vnv). (F) A maximum-likelihood tree shows ABSs of Acanthamoeba MBPs form a cluster, which is distinct from those of bacteria. These results strongly suggest the Acanthamoeba ABS derived by HGT, although the precise bacterium cannot be identified. (G) A Western blot shows an E. coli maltose-binding protein (EcMBP) fused to ABS binds so strongly to a mannose-agarose resin that it cannot be eluted with excess α-methyl-mannose but can only be released with SDS. (H and I) The negative control EcMBP alone fails to bind to the mannose-agarose resin, while the positive control ConA is eluted from the mannose-agarose resin with excess α-methyl-mannose. (J–L) Confocal microscopy shows EcMBP-ABS and ConA (positive control) each bind to HCLE cells labeled with an anti-tubulin antibody and DAPI, while EcMBP alone (negative control) fails to bind to HCLE cells. (M and N) EcMBP-ABS does not bind to Acanthamoeba cysts or trophozoites. (O) Double labels with Jonah-1-mCherry and Luke-2-GFP or laccase-1 and Leo-A-GFP show incubation with excess α-methyl-mannose has no effect on cyst wall formation. Scale bars for panels J–O are each 5 µm.

Fig 6

Anti-ManBD rAbs efficiently detect DAPI-labeled trophozoites of all 11 isolates of Acanthamoeba. (A) Confocal micrographs show rAbs to EcMBP-ABS, renamed here ManBD, binds to the surface of DAPI-labeled trophozoites of the Neff strain of Ac (high power on left and low power on right). (B) Binding of anti-ManBD rAbs to Neff trophozoites is also easily visualized with conventional immunofluorescent microscope (high power on left and low power on right). (C) Pairs of high power (left) and low power (right) confocal micrographs show anti-ManBD rAbs bind to trophozoites of 10 other isolates Acanthamoeba, which were used for binding anti-cyst antibodies in Fig. 2 and 3. (D) Counts of trophozoites in low-power micrographs show that >90% of DAPI-labeled trophozoites were detected by rAbs to the ManBD. (A and B) Scale bars for high-power micrographs are 5 µm and for low-power micrographs are 50 µm.