The Metallochaperone Encoding Gene hypA Is Widely Distributed among Pathogenic Aeromonas spp. and Its Expression Is Increased under Acidic pH and within Macrophages

Metallochaperones are essential proteins that insert metal ions or metal cofactors into specific enzymes, that after maturation will become metalloenzymes. One of the most studied metallochaperones is the nickel-binding protein HypA, involved in the maturation of nickel-dependent hydrogenases and ureases. HypA was previously described in the human pathogens Escherichia coli and Helicobacter pylori and was considered a key virulence factor in the latter. However, nothing is known about this metallochaperone in the species of the emerging pathogen genus Aeromonas. These bacteria are native inhabitants of aquatic environments, often associated with cases of diarrhea and wound infections. In this study, we performed an in silico study of the hypA gene on 36 Aeromonas species genomes, which showed the presence of the gene in 69.4% (25/36) of the Aeromonas genomes. The similarity of Aeromonas HypA proteins with the H. pylori orthologous protein ranged from 21−23%, while with that of E. coli it was 41−45%. However, despite this low percentage, Aeromonas HypA displays the conserved characteristic metal-binding domains found in the other pathogens. The transcriptional analysis enabled the determination of hypA expression levels under acidic and alkaline conditions and after macrophage phagocytosis. The transcriptional regulation of hypA was found to be pH-dependent, showing upregulation at acidic pH. A higher upregulation occurred after macrophage infection. This is the first study that provided evidence that the HypA metallochaperone in Aeromonas might play a role in acid tolerance and in the defense against macrophages.


Introduction
Metal ions are essential for the correct function of microbial biological processes [1]. In fact, many proteins contain metal ions bound directly to their amino acid chains by histidine or cysteine residues as cofactors. Particularly, metalloenzymes such as nitrogenases, ureases or hydrogenases are abundant types of metalloproteins that catalyze numerous metabolic and enzymatic reactions [2][3][4]. The synthesis of these metalloenzymes consists of complex processes that require a set of accessory proteins. In this context, metallochaperones play a key role in bacterial metal homeostasis ("metallostasis") since they are involved in the acquisition and transfer of metals [4][5][6]. Many lines of evidence indicate an important role of metallostasis in the host-pathogen interaction [1,6,7]. For instance, during infection the host limits the availability of essential metals, inactivating metal-dependent processes of the bacterial pathogen, which compensates for this limitation by producing metallochaperones, among other proteins.
For evaluation of urease activity, the bacteria were grown in Urea Agar Base media (with a yellow-orange color) at 37 • C for 24 h and the change of color of the slant indicated a positive reaction. A strain of Salmonella sp. was used as a negative control and one of Proteus sp. as a positive control. The strains used in this study belong to our laboratory collection (Microbiology unit, University Rovira I Virgili)

In Silico Search of hypA and ure Genes in the Aeromonas Genomes
For identification of hypA sequences in Aeromonas species, an initial text-based search was conducted on UniProtKB from Uniprot database (https://www.uniprot.org/uniprot/). As a result, the hypA deduced amino acid sequence from Aeromonas hydrophila CECT 839 T (A0KL76) was found. The corresponding nucleotide sequence of this strain (ABK37593.1) was used as query for BLAST search on 36 genomes of Aeromonas spp. type strains deposited in the NCBI database (https://blast.ncbi. nlm.nih.gov/Blast.cgi) to identify hypA orthologues sequences. To investigate whether the presence of hypA could be a species-specific characteristic, we extended our analysis to other available genomes of non-type strains (n = 110). These genomes were verified with the Average Nucleotide Identity (ANI) using the online tool OrthoANI or with the analysis of the rpoD gene [27,28]. For identification of urease genes in the Aeromonas genomes, another BLAST search was performed using as query the sequences of the genes: ureA, ureB, ureI, ureE, ureF, ureG and ureH extracted from the genome of H. pylori strain HPAG1 (annotated in the NCBI database).

Protein Sequence Analysis and 3D-Structure Prediction
To determine sequence conservation of hypA in Aeromonas, a comparison of HypA proteins among the 25 Aeromonas species and one of E. coli (strain E24377A) and H. pylori (strain HPAG1) was assessed by multiple alignments, using the CLUSTALW algorithm via MegAlign. Phylogenetic relationships among sequences were depicted in a phylogenetic tree constructed with MEGA6 using the Neighbor-joining and Maximum-likelihood algorithms. In addition, the prediction of 3D monomeric and dimeric HypA protein structure and the comparative analyses of this protein with those of A. hydrophila (CECT 839 T ) and E. coli (E24377A), was done using the Swiss-model online tool (https://swissmodel.expasy.org/).

Cell Line Culture, Infection and Induction Experiments
The cell line J744A.1 from mouse BALB/C monocyte macrophages was used for the infection experiments with the six Aeromonas type strains. The macrophages cells were maintained in adhesion in Dulbecco's Modified Eagle's Medium (DMEM; Biowest, Nuaillé, France) (pH = 8) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) plus 1% penicillin-streptomycin solution (P/S; Biowest, Nuaillé, France) at 37 • C and 5% CO 2 . Prior to infection, cells were seeded in tissue culture plates (1 × 10 6 cells/mL) containing serum-free DMEM without antibiotics (serum-starvation conditions) for 18 h. The macrophages J774A.1 were infected with the six Aeromonas type strains grown in serum-free DMEM without antibiotics at a multiplicity of infection (MOI) of 5. In addition, bacteria were seeded onto tissue culture plates in serum-free DMEM without antibiotics at alkaline pH (pH = 8) or acidic pH (pH = 4.5) adjusted with an HCl solution followed by filtration to remove any precipitate. Co-cultures were incubated at 37 • C and 5% CO 2 up to 4 h for gene expression analyses.

RNA Extraction and Quantitative RT-PCR
Total RNA was isolated from logarithmic-phase Aeromonas cultures using TRIzol ® Reagent (Invitrogen, Carlsbad, CA, USA) as previously described [29]. RNA quality and integrity were confirmed spectrophotometrically using Nanodrop 2000, calculating the 260/280 and 260/230 ratios. The cDNA was transcribed from RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA) according to the manufacturer's instruction. Quantitative Real-Time PCR was performed in duplicate using Real-Power SYBR ® green PCR Mastermix (Applied Biosystems ® , Waltham, MA, USA) in 10 µL total PCR reaction mixture on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). The thermal cycling conditions were: 94 • C for 5 min, followed by 45 cycles of 30 s at 94 • C, 30 s at 60 • C, 30 s at 72 • C, and finally, 20 s at 80 • C. The threshold cycle (Ct) was automatically determined by the StepOne Software v2.0 (Applied Biosystems) to calculate the relative expression of the tested gene (hypA) using as reference the 16S rRNA housekeeping gene, as previously described [30]. Relative gene expression levels and fold change expression were estimated using 2 −∆∆Ct method [31]. The specific primer pairs for the PCR amplification of hypA and 16S rRNA were designed by using consensus nucleotide sequences and Oligo Primer Analysis Software v. 7 (Table 1). Experiments were performed in triplicate using three independently prepared bacterial growth cultures obtained on three different days.

Statistical Analysis
All experiments were performed in triplicates and significant differences were determined using Student's two-tailed t-test calculated using GraphPad Prism 6.0 (GraphPad Software, CA, USA). p values ≤ 0.05 were considered statistically significant (*).

Identification of hypA Gene in Aeromonas Species
The results of the in silico search using the hypA sequence (339 bp) of A. hydrophila CECT 839 T as template showed that hypA was present in 69% (25/36) of investigated genomes of type strains belonging to the Aeromonas species shown in Table 2. Extended analyses on 108 additional genomes from those 36 Aeromonas species available in NCBI database, allowed investigation of whether hypA is a strain or a species-specific character (Table S1). We detected hypA gene sequences in 83% (122/146) of all genomes analyzed. In most species analyzed, concordance between strains regarding the presence/absence of hypA in their genomes is observed. Although it is interesting to mention that A. caviae, A. schubertii and A. media showed a discrepancy, since some of the strains have hypA and others do not (Table S1).

Sequence Analyses of hypA Proteins Shows Specific Motifs Associated to Metal Binding
The specific metal-binding motifs consisting of N-terminal MHE motif for Ni-binding and two consecutive cysteine motifs CxxCnCPxC for Zn-binding, previously reported in the HypA proteins of E. coli and H. pylori, were also observed in the Aeromonas spp. protein sequences in Figure 1A. The three-dimensional predicted structures of HypA proteins of A. hydrophila CECT 7996 T and E. coli show high similarity among them ( Figure 1A). Indeed, monomeric and dimeric predicted proteins of the latter species displayed the characteristic α-helices (α1 and α2) and a β-sheet (long β1, β2, and β6 and short β3, β4, and β5) ( Figure 1B). consecutive cysteine motifs CxxCnCPxC for Zn-binding, previously reported in the HypA proteins of E. coli and H. pylori, were also observed in the Aeromonas spp. protein sequences in Figure 1A. The three-dimensional predicted structures of HypA proteins of A. hydrophila CECT 7996 T and E. coli show high similarity among them ( Figure 1A). Indeed, monomeric and dimeric predicted proteins of the latter species displayed the characteristic α-helices (α1 and α2) and a β-sheet (long β1, β2, and β6 and short β3, β4, and β5) ( Figure 1B).

Conservation and Phylogenetic Relationships of hypA in Aeromonas
In silico HypA is the similarity of the in silico-translated amino acid sequences of HypA between the Aeromonas species ranged between 86% and 100%. As expected, when comparing the Aeromonas HypA with those from E. coli and H. pylori the similarity was significantly lower, 41-45% and 21-26% respectively ( Figure 1A). As observed in the phylogenetic tree, HypA proteins were highly conserved among the 25 Aeromonas species. Two groups of species, one formed by A. aquatilis, A. sobria and A. saranellii and the other with the species A. encheleia and A. aquatica had identical hypothetical protein sequence (Figure 2 and Figure S1). Thus, in conclusion, we can state that HypA was highly conserved within the genus Aeromonas.
In addition, it is important to mention that there are many identified hypA encoded proteins across different genus and bacterial species, which provide evidence of their conservation throughout evolution (Table S2). However, little is known about the biological function of HypA proteins in the vast majority of these species.

Transcriptional Regulation of hypA under Different pH-Condition and Macrophage
The expression patterns of hypA determined under stressful pH conditions in the most prevalent clinical species (A. hydrophila, A. caviae, A. dhakensis and A. veroni) and in two species frequently associated with fish diseases (A. salmonicida and A. piscicola) by qRT-PCR are shown in Figure 3A. All Aeromonas species displayed a similar relative expression of hypA in alkaline conditions (pH 8) ( Figure 3A). Nevertheless, the expression of hypA was significantly higher (p < 0.05) under acid condition (pH 4) in comparison to alkaline condition (pH 8), displaying a greater upregulation in the most prevalent clinical species (Figure 3A,B). Furthermore, given that the phagosome of macrophages becomes acid upon phagocytosis of pathogens, we evaluated the expression of hypA during Aeromonas infection. The results showed that Aeromonas upregulates hypA in response to phagocytosis, displaying a significantly higher expression of the metallochaperone during infection than in control (alkaline media) or in vitro acid exposure (p < 0.05) ( Figure 3A,C). Although transcriptional regulation of hypA seems to depend on pH or infection condition, the statistical analysis revealed strain-related differences. The most clinically prevalent species showed a significantly higher upregulation of hypA under acid exposure and during infection than species considered environmental or more related to fish disease ( Figure 3B,C). Significant differences in gene induction under acid exposure were observed among the most prevalent clinical species, except between A. hydrophila and A. veronii ( Figure 3B). Additionally, there were significant differences in the group of clinically prevalent species, with A. veronii showing notably lower induction of hypA when compared with A. hydrophila, A. caviae and A. dhakensis during macrophage infection (p < 0.05) ( Figure 3C).
With the purpose of understanding how Aeromonas adapts to low pH environments, we determined the pH variations of the medium during in vitro growth or during infection of macrophages with the species under study. No significant changes in pH were observed when strains where grown in DMEM at pH 4 or pH 8 with the only exception of A. hydrophila (CECT 839 T ). When the latter strain was incubated in DMEM at pH 4 there was an increase of pH that reached up to 7.5 and that was visually evident by the changing color of the DMEM media, that functions as a pH indicator ( Figure 4A,C). This basification of the medium or other pH changes were not observed for other species, neither for the infected or uninfected macrophages ( Figure 4C).

Urease Activity and Urease Genes in Aeromonas Species
HypA is also involved in the urease maturation which facilitates the survival of bacteria in the human gastric mucosa neutralizing the acidic environment [17]. Therefore, we have evaluated the ability of Aeromonas to hydrolase urea by determining urease activity using a biochemical method. None of the strains assayed produced ureases because no color change in the media from light orange to magenta was observed when compared with the positive urease control (Proteus sp.). In addition, the battery of proteins associated with urease activity was not found in the genome of the six strains under study [32]. This is consistent with the absence of the urease genes in Aeromonas sp., which is inferred from our in silico search of all available genomes of the Aeromonas genus.

Discussion
The relevance of bacterial metal homeostasis is related to the essential role that metals play for survival in different environments, including the context of host-pathogen interaction during the infection processes [33]. In the last years, there has been significant progress in the knowledge of how metallochaperones bind metal ions, recognize the target proteins and facilitate metal transfer [4,6]. The HypA metallochaperone has been associated with [NiFe] hydrogenase and urease maturation and is considered a relevant protein for adaptation to acidic environments of pathogenic bacteria like H. pylori and E. coli [17][18][19]. Furthermore, the HypA metallochaperone participates in the defense against oxidative environments [20].
The present work is the first to address the study of the HypA metallochaperone in the genus Aeromonas. Our results suggest that hypA genes in certain species, like A. hydrophila, A. dhakensis, A. veronii and A. taiwanensis among others, are widely conserved among their strains. However, other species like A. caviae, A. schubertii and A. media showed strain-level variants, in which some strains from the same species contain hypA and others do not. Such lack of uniformity in hypA presence in well-represented species, as A. veroni, would indicate within-species microevolutionary changes that could result from environmental adaptation [34]. On the other hand, in our study some species are poorly represented, and additional analyses should be performed in order to give precise conclusions. Overall these data indicate that hypA tends to be conserved within species although cannot be considered strictly a species-specific character.
The Aeromonas HypA protein sequences showed to be moderately similar to those described in E. coli and H. pylori. However, they display the characteristic N-terminal MHE motif for Ni-binding and two consecutive cysteine motifs CXXCnCPXC for Zn-binding, also conserved in the tow human pathogenic bacteria mentioned [13,14,16,17]. In addition, the three-dimensional structure predicted for all proteins showed to be highly similar among the Aeromonas species and the other two human pathogenic bacteria, confirming that it is a metallochaperone [15,16,35].
Although the principal role of HypA has been typically associated with the maturation of hydrogenases, in the last years it has been demonstrated that HypA could play a role in the maturation of ureases. These metalloenzymes are involved in the survival of pathogenic bacteria in an acidic environment. For instance, bacterial ureases are involved in the survival of H. pylori in the human stomach at acidic pH [17][18][19]. In E. coli this function is carried out by other enzymes i.e., hydrogenases [17,21,22]. The fact that 80% of infections caused by Aeromonas are gastrointestinal diseases indicates an adaptation of these bacteria to acid environments of the gastrointestinal tract [25,26]. Additionally, a previous study hypothesized that urease activity may contribute to acid tolerance in some A. caviae strains, facilitating bacterial survival during infection, as occurs in Yersinia enterocolitica [36]. However, Aeromonas species usually have been described as urease negative [37]. Consistent with these previous data we observed in our study that all strains were urease negative. However, the expression study demonstrates that hypA is upregulated in acidic pH. A reasonable explanation could be that hydrogenases, but not ureases, would be involved in acid resistance in Aeromonas, as occurs in E. coli [18,22,23]. In addition, our results showed that A. hydrophila CECT 839 T alkalinize the medium during acid exposure. One feasible explanation for this phenomenon would be that A. hydrophila has a higher tolerance to acids as a consequence of an enzymatic pH shifting, which allows a better survival. Although additional analysis including more strains should be performed to determine if this is a specific characteristic of this species. Therefore, it would seem plausible to affirm that HypA could be associated with acid tolerance in Aeromonas species.
Numerous studies provide insights into the relevance of redox signaling and reactive oxygen species (ROS) production as defense mechanisms against pathogens [38,39]. Moreover, bacterial infections also can induce oxidative stress, which contributes to increase the rates of DNA mutations in the host. For instance, H. pylori produce superoxide anion in order to counteract the toxic effect of the ROS produced in the human stomach, which contributes even more in the development of gastric cancer [40]. Considering that the immune system generates ROS as defense mechanism against pathogens after phagocytosis by macrophages [41,42], resistance to acidic environments can be of great advantage for pathogens. Indeed, some studies have emphasized a strong relationship between deficiency of ROS production and susceptibility to microbial infection [43]. In these contexts, the discovery of the chaperone Hsp33 and its role in protecting cells against the deleterious effects of reactive oxygen species [44][45][46], reinforces the hypothesis of the important role of redox regulation during bacterial colonization [47]. In our study, the results showed that the Aeromonas metallochaperone HypA was upregulated after phagocytosis of macrophages, which is in line with the previous works [44][45][46]. Therefore, considering that hydrogenases participate in oxidative stress defense [20] it is very possible that HypA also contributes to the defense against ROS produced by macrophages in the phagocytic process.
According to the literature, 96.5% of the clinical Aeromonas strains correspond to four species: Aeromonas caviae (29.9%), Aeromonas dhakensis (26.3%), Aeromonas veronii (24.8%) and Aeromonas hydrophila (15.5%). However, other species usually associated with a fish disease like A. salmonicida have been isolated from human infections [25]. Our results showed a higher upregulation of the hypA after infection with the most prevalent clinical species, independently of the condition. Specifically, one strain used in the study (A. hydrophila CECT 839 T ) was isolated from milk although its virulence was previously demonstrated [48]. Otherwise, the expression of the hypA after infection with the strain A. salmonicida CECT 894 T , described as a pathogenic for fish, was lower. These results could be associated with that possibility of a more important role of hypA in human health compared to animals, as is the case with other human pathogens as E. coli and H. pylori. However, further studies with a higher number of strains to better corroborate this hypothesis are needed.
In conclusion, our results suggest that HypA could play a role in the survival of Aeromonas in acidic environments and in defense against macrophages, although the exact mechanism remains unclear, but the possible role of this metallochaperone in Aeromonas sp. virulence is evident.

Conclusions
This study reports for the first time the distribution of orthologous sequences coding for the metallochaperone HypA in bacterial genomes of the genus Aeromonas and their deduced protein structure. Interestingly, HypA was present in 69.4% of Aeromonas species showing high similarity among the species (%). Metallochaperones are relevant proteins in the host-pathogen interaction. Thus, the present study suggests a possible role of HypA in bacterial survival in acidic environments, as well as in the defense against ROS produced by macrophages. In addition, it may promote future studies to confirm and better understand the function of this metallochaperone in the survival of species from the genus Aeromonas.