Revisiting the Ancylostoma Caninum Secretome Provides New Information on Hookworm–Host Interactions

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil‐transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anemia and other complications. These parasites release a variety of molecules known as excretory/secretory products (ESPs) that are involved in many different biological processes that govern parasite survival. Using a combination of separation techniques such as SDS‐PAGE and OFFGEL electrophoresis, in combination with state‐of‐the‐art mass spectrometry we have reanalyzed the dog hookworm, Ancylostoma caninum, ESPs (AcESP). We identified 315 proteins present in the AcESP, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (110 of the 315 proteins), and the most abundant constituents of AcESP are homologues of the tissue inhibitors of metalloproteases (TIMP) family. Interestingly, we identified new homologs of well‐known vaccine candidates and immunomodulatory proteins. This study provides novel information about the proteins secreted by A. caninum, and constitutes a comprehensive dataset to study the proteins involved in host–hookworm interactions.


DOI: 10.1002/pmic.201700186
and chronic infections result in irondeficiency anemia and even physical and intellectual retardation in young children. [3] Adult hookworms live in the intestine of vertebrate hosts where they feed on blood, and constantly release products into their surrounding environment through excretion and secretion mechanisms (excretory/secretory products, ESPs). The ESPs contain proteins that facilitate a parasitic existence, notably penetration of and migration within a host, feeding on host tissues, and evasion of the host immune response. [4] In addition, recently, hookworm ESPs have been shown to contain immunoregulatory properties that can protect mice against inflammatory diseases such as inflammatory bowel diseases and asthma. [5][6][7][8] Due to the difficulty in obtaining Necator americanus adult worms from the human host, the dog hookworm Ancylostoma caninum has been extensively used as a model to study hookworm infections. The first proteomic characterization of the ESPs produced by A. caninum (AcESP) was performed by Mulvenna et al. [9] ; however, herein we revisit this data since the A. caninum genome was not available at the time and the sensitivity of mass spectrometers has improved dramatically since this last study was conducted.
A total of 300 A. caninum adult worms were obtained from the small intestine of five fresh cadaver dogs that had been naturally infected. Worms were divided into two different batches and incubated in 5× substrate (Dulbecco's PBS (DPBS) (+) CaCl 2 (+) MgCl 2 , 5% antimycotic/antibiotic, 1% Glutamax) for 2 h at 37°C and 5% CO 2 to reduce bacterial contamination. Hookworms were then transferred to 2× substrate and incubated for a further 24 h at 37°C and 5% CO 2 at a rate of 50 worms per 25 mL of media.
A total of 50 μg of AcESP from batch 1 was separated by SDS-PAGE and 18 bands were excised from the gel, reduced using dithiothreitol (DTT), alkylated with iodoacetamide (IAM), and digested with trypsin overnight as described previously. [9] One hundred (100) micrograms of AcESP from batch 2 was reduced and alkylated using DTT and IAM, respectively followed by trypsin digestion overnight. Peptides were separated using an OFFGEL fractionator as previously described. [9] All samples were desalted using C18 ZipTips after SDS-PAGE or OFFGEL separation. Peptides were analyzed using a Shimadzu Prominance Nano HPLC coupled to an AB SCIEX Triple TOF+ 5600 mass spectrometer and processed using the software Analyst TF 1.6.1. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [10] partner repository with the dataset identifier PXD006511 and https://doi.org/10.6019/PXD006511. Database searches were performed against a database consisting of the A. caninum genome and proteins from the common Repository of Adventitious Proteins (cRAP, http://www.thegpm.org/crap/) with Mascot using Mascot Daemon (v.2.5.1, Matrix Science) and X! Tandem (v.2015.12.15.2) and Comet (v.2016.01 rev.2) using PeptideShaker (v.1.11.0). [11] Annotation of proteins was performed using Blast2GO, [12] Pfam, [13] PANTHER, [14] and InterProScan. [15] A total of 237 and 289 proteins were identified with two or more peptides and FDR < 1% using PeptideShaker and Mascot (Supporting Information, Tables S1 and S2), respectively, from which 211 were common between both search programs, while 26 and 78 were uniquely found by PeptideShaker and Mascot, respectively, resulting in a final quantity of 315 AcESP proteins (Supporting Information, Table S3). A total of 67 out of the 105 proteins identified by Mulvenna et al. [9] were also found in the present study.
The top three most abundant proteins found by Mascot ( Table 1) were ANCCAN 13497 (a previously described tissue inhibitor of metalloproteases; TIMP [16] ), ANCCAN 25071 (a hypothetical protein), and ANCCAN 19759 (a sperm-coating protein; SCP, also called SCP/Tpx-1/Ag5/PR-1/Sc7 domain containing proteins; SCP/TAPS). The top three proteins found by X! Tandem and Comet using PeptideShaker ( Table 2) were ANC-CAN 03259 and ANCCAN 01699, two SCP proteins, and AN-CCAN 13497 a TIMP (also found by Mascot in the top three most abundant proteins). TIMP proteins are a multifunctional family of inhibitors of matrix metalloproteases associated with different functions in eukaryotic systems such as tissue remodeling, extracellular matrix turnover, cell proliferation, and angiogenesis among others. [17] However, in parasites, it has been previously suggested that TIMP-like proteins might not be functioning as matrix metalloproteases inhibitors. [18] The TIMP-like protein ANCCAN 13497 (previously identified as Ac-TMP-1 [16] ) was already identified by Mulvenna  (and only TIMP-like) protein in the AcESP. [9] Interestingly, other A. caninum TIMP-like protein, the renamed Anti-Inflammatory Protein-2 (AIP-2), has been shown to be a potent immunomodulatory protein that suppresses airway inflammation in a mouse model of asthma. [7] AIP-2 has extensive homology with other TIMPs found in AcESP, including the highly abundant AN-CCAN 26655 and ANCCAN 24968, suggesting that these two proteins could have similar immunomodulatory properties. Four different TIMPs are present in AcESP, and are highly abundant based on the emPAI and spectral count. The SCP proteins are highly represented in the infective larval stage of hookworms, where a role in larval penetration and infection has been hypothesized (reviewed by [19] ). They have also been speculated to be involved in immunomodulation. [20] For instance, an A. caninum SCP-like protein (known as Neutrophil Inhibitory factor; NIF) is able to inhibit neutrophil function and oxidative stress. [21] Although we did not find this protein in the adult secretions, we have found two SCPs having extensive homology with the NIF sequence deposited in NCBI (accession number AAA27789.1): ANCCAN 04194 (88% identity and E-value = 6.9 × 10 −89 ) and ANCCAN 22933 (94.3% identity and E-value = 1.5 × 10 −65 ). This could refer to a misannotation of the genome, since a blast search against the A. caninum genome using the deposited NIF sequence does not return any sequence with 100% homology. SCP-related proteins (including SCPs, SCP-homolog proteins, and SCP-like proteins) contributed to 110 out of 315 (31%) of the overall protein families and were by far the most highly represented family of proteins in the AcESP (Figure 1). These results suggest that the SCP family might play a key role in orchestrating a parasitic existence and modulating immune response of the host, and further studies should focus on this family of proteins.
Among the most represented protein domains in AcESP was the metallopeptidase family M13 (11 proteins), the transthyretinlike family (ten proteins), astacin metalloproteases (seven proteins), and glutathione-s-transferases (GSTs; six proteins). The role of peptidases in the secretome of helminths has been linked to important roles in parasitism [22] ; however, the roles of transthyretin-like proteins are still unknown (despite their abundance in nematodes in particular). Astacins are a family of metallopeptidases highly abundant in the secretome of helminths. [23] Indeed, the human hookworm N. americanus has 82 astacin-encoding genes, [24] and astacins are abundantly represented in ESPs of the rat hookworm Nippostrongylus brasiliensis [25] and other nematodes. [26] The role of astacins is not fully understood, although, in nematodes, they have been shown to participate in host invasion and parasite development. [27] It is also noteworthy to highlight the presence of GSTs in the AcESP proteome. Na-GST-1, a GST secreted by adult stage N. americanus that is thought to play a role in feeding by detoxifying the free heme produced after hemoglobin ingestion, is being currently tested as a vaccine against N. americanus. [28] Other molecules that have been tested as vaccines against hookworms include aspartic proteases and cysteine proteases (reviewed by [29] ), which are protein families also represented in our study (Figure 1). In the present study we found five different cysteine proteases (AN-CCAN 06649, ANCCAN 06644, ANCCAN 30567, ANCCAN 06619, and ANCCAN 06647) and four aspartic proteases (ANCCAN 18339, ANCCAN 13546, ANCCAN 29459, and AN-CCAN 25067). Thus, it is tempting to speculate that the GSTs, cysteine proteases and aspartic proteases identified in the present study could be potential vaccines against the dog hookworm.
In the present study, we have reanalyzed the protein constituents of AcESP in order to gain a more comprehensive snapshot of the hookworm secretome and how this impacts on host-pathogen interactions. We have identified almost three times as many proteins as previously reported in the ESP of this important parasite. In addition, new proteins of interest with potential (based on their homology to other molecules) as both novel immunoregulatory biologics and vaccine candidates have been identified, and clearly warrant future exploration.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.