1. Introduction
Acinetobacter baumannii is an opportunistic human pathogen, found predominately in hospitals and aged-care facilities [
1].
A. baumannii can cause a wide variety of diseases including pneumonia, wound and burn infections, meningitis, and urinary tract infections. While most
A. baumannii infections are nosocomial, community-acquired
A. baumannii infections do occur in immune-compromised individuals [
2].
A. baumannii is attracting significant attention for its increasing antimicrobial resistance, and the global spread of strains resistant to all available antibiotics is imminent. Carbapenem-resistant
A. baumannii has been placed at the top of the World Health Organization (WHO) critical pathogens list, being in urgent need of new antibiotics [
3].
A. baumannii is believed to employ a “persist and resist” strategy, wherein the bacterium adapts to unfavourable conditions by processes including biofilm formation, survival from dissociation, and resistance to a range of antimicrobial stresses, including biocides, antibiotics, and metals [
4,
5,
6].
Metals, such as copper, are used as antimicrobials in healthcare settings and agriculture [
7,
8]. Further, copper has been shown to be recruited by macrophages and neutrophils for its antibacterial activity within the host [
9,
10,
11,
12]. Copper toxicity is primarily associated with increased susceptibility to oxidative stress and mismetallation of non-copper metalloproteins [
13]. To counteract host-mediated or environmental copper intoxication, bacteria can express a wide range of efflux systems to maintain metal ion homeostasis. These can be classified into three main families, the heavy metal efflux (HME) family (which is a subfamily within the resistance-nodulation-cell division (RND) superfamily), the P-type ATPase family, and the cation diffusion facilitator (CDF) family.
The HME subfamily of transporters facilitates export of metal ions from the cytoplasm and/or periplasm to the extracellular environment, via a proton-antiport driven process [
14]. One example of this family is the copper efflux system CusABC, in
Escherichia coli [
15]. The members of the HME family of exporters consist of three proteins, namely, an inner membrane protein (CusA), a periplasmic adaptor protein (CusB), and an outer membrane protein (CusC). Periplasmic copper is delivered to the CusABC system by CusF, a periplasmic copper chaperone [
15]. The P-type ATPases are another large family of transport proteins, which are dependent on adenosine triphosphate (ATP) hydrolysis for activity [
16]. In Gram-negative bacteria, this family of transporters has been associated with the unidirectional transport of ions, in either an import or efflux capacity, across the cytoplasmic membrane. One of the most well-characterised P-type ATPases is CopA from
Legionella pneumophila, which effectively exports copper across the cytoplasmic membrane [
17]. Members of the CDF family of transport proteins are exclusively involved in metal ion export. Although zinc, cadmium, and cobalt are the primary substrates of CDF proteins [
18], studies have shown that some members of this family can provide protection to other metals, including copper [
19]. CDF transporters are antiporters and export divalent metal ions in exchange for monovalent cations (H
+ or K
+). Bacterial CDFs are ubiquitous, with YiiP from
E. coli being a well-characterised representative [
20].
In addition to the three main families of metal ion efflux proteins, there are other copper resistance systems found in Gram-negative bacteria, such as the periplasmic multi-copper oxidase (e.g., CueO from
E. coli), that oxidizes Cu
1+ to Cu
2+ [
21]. Further, CopB, an outer membrane protein, is involved in periplasmic copper resistance; however, exactly how this is mediated remains unknown [
22]. Also involved in dealing with periplasmic copper stress is the inner membrane protein CopD, which is predicted to import copper into the cytoplasm, a process aided by the periplasmic CopC copper-binding protein [
23,
24,
25]. Cytoplasmic copper stress is sensed by the MerR-family regulator CueR, which has been shown to be responsible for regulation of, for example,
copA in
E. coli [
26]. In contrast, periplasmic copper stress sensing is believed to be mediated by CopRS, a two-component regulatory system [
27].
Recently, candidates for many of the copper resistance mechanisms described here were identified in
A. baumannii [
6,
28]. However, their genetic organisation, regulation, contribution to copper tolerance, and in vivo virulence remain to be determined. Here, we provide a comprehensive overview of the
A. baumannii copper resistome and identify the P-type ATPase CopA as the primary cytoplasmic copper stress resistance determinant. Subsequently, our analyses of a
copA mutant derivative show that CopA represents a key modulator of oxidative stress resistance and colonisation of the host’s respiratory tract in a murine model of
A. baumannii infection.
3. Discussion
In this study, we examined
A. baumannii copper resistance by bioinformatically analysing the putative cytoplasmic and periplasmic copper homeostasis mechanisms. This study has provided new insight into the relative roles of the CueR and CopRS regulators in
A. baumannii copper resistance. We also found that various
A. baumannii copper resistance mechanisms were positioned on mobile genetic elements, including the ABUW_3320−3327 cluster, and at least one additional cluster encoded on plasmid pC13-2, which shares a high level of homology to ABUW_3320–3327 [
31]. Our work has emphasised that
A. baumannii uses a combination of copper resistance systems that are, at least in part, distinct to that observed in other previously studied Gram-negative bacteria, such as
E. coli and
P. aeruginosa. The Cus HME transport system provides
E. coli with copper resistance during anaerobic conditions [
32]; therefore, the lack of a commonly expressed copper HME efflux system in
A. baumannii may be due to it being a strictly aerobic bacterium. Further, the absence of CopR binding sites in the
pcoAB cluster of
A. baumannii, and positioning of
copRS on the accessory genome, has highlighted that regulation of PcoAB-mediated periplasmic copper resistance in
A. baumannii requires further examination.
We also showed, for the first time, that the chromosomally-encoded, and highly conserved, P
1B-1 ATPase CopA provides a high level of copper resistance and protects the cell against oxidative stress. The in vivo significance of our findings was shown by impairment of the
copA::T26 mutant in the bronchoalveolar lavage (BAL), nasal wash and, to a lesser extent, the lungs. This may be due to copper being an important antimicrobial at mucosal surfaces. An alternative explanation for the difference between the wild-type and
copA::T26 mutant in colonisation may centre around differential clearance by phagocytic cells. Phagocytic cells have been previously demonstrated to harness the antimicrobial activity of copper to kill bacterial pathogens [
28]. Accordingly, the differences seen between the wild-type and
copA::T26 mutant strains may be a result of their resistance to copper-prosecuted phagocytic killing. Interestingly, the analysis of a putative copper acquisition system, OprC, has previously revealed the significance of copper acquisition in
A. baumannii virulence, including colonisation of lung tissue [
33]. Combined, this highlights the importance of both import and efflux to provide
A. baumannii with a functional copper homeostasis system during infection.
Through comparative genomics and subsequent copper susceptibility analyses, we have previously speculated that the ABUW_3320–3327 cluster does not play a critical role in copper resistance, as most strains of
A. baumannii were affected by copper to a similar extent, regardless of the presence of this cluster [
28]. In line with these findings, our analyses of a
cueA::T26 P
1B-1 ATPase mutant (ABUW_3325) did not reveal a role in copper tolerance. Considering the role of all other members of the ABUW_3320–3327 cluster in dealing with periplasmic copper stress, this observation is not surprising, as CueA-mediated transport of copper back into the periplasm following its removal by CopD would be unanticipated. A factor that should be considered when comparing the functional relevance of
A. baumannii CopA and CueA P
1B-1 ATPases is the presence of the copper chaperone
copZ in the same genetic cluster as
copA, but not
cueA, which may contribute to their relative copper efflux efficiencies.
Further bioinformatic analyses revealed that CueA of
A. baumannii belongs to a distinct group of P
1B-1 ATPases, which we denoted the “histidine-rich” subgroup. P
1B-1 ATPases with an elevated number of histidine residues have been previously described in the analysis of P-type ATPases from the Rhizobiales order, which includes nitrogen-fixing plant symbionts, such as the
Sinorhizobium species, and the human pathogens of the
Bartonella species [
34]. Although none of the histidine-rich P
1B-1 ATPases from
Sinorhizobium species have been functionally characterised, the roles of LpCopA and SilP in metal resistance illustrate that the histidine-rich P
1B-1 ATPases can fulfil important roles. Examination of the key metal-binding residues, as identified in the LpCopA structure, did not reveal any differences between members of the classical and histidine-rich P
1B-1 ATPases, with the sole exception of LpCopA M711 [
17]. We found M711 to be distinct between various species, even within members of the two P
1B-1 subgroups, which indicates M711 may not play a critical role in the final copper extrusion step by LpCopA.
Overall, this study highlights the significance of cytoplasmic copper toxicity in A. baumannii and how it overcomes this to allow expression of its full host-colonisation potential. Further, we have provided novel insights into the variation seen within bacterial P1B-1 ATPases. Our analyses also show that CopA and CueA are not functionally redundant, as predicted by bioinformatic analyses.