Iron Acquisition Mechanisms and Their Role in the Virulence of Acinetobacter baumannii

ABSTRACT

Iron is an essential element for survival of most organisms. One mechanism of host defense is to tightly chelate iron to several proteins to limit its extracellular availability. This has forced pathogens such as Acinetobacter baumannii to adapt mechanisms for the acquisition and utilization of iron even in iron-limiting conditions. A. baumannii uses a variety of iron acquisition strategies to meet its iron requirements. It can lyse erythrocytes to harvest the heme molecules, use iron-chelating siderophores, and use outer membrane vesicles to acquire iron. Iron acquisition pathways, in general, have been seen to affect many other virulence factors such as cell adherence, cell motility, and biofilm formation. The knowledge gained from research on iron acquisition led to the synthesis of the antibiotic cefiderocol, which uses iron uptake pathways for entry into the cell with some success as a novel cephalosporin. Understanding the mechanisms of iron acquisition of A. baumannii allows for insight into clinical infections and offer potential targets for novel antibiotics or potentiators of current drugs.

INTRODUCTION

The Acinetobacter genus contains many species known for being opportunistic pathogens; however, Acinetobacter baumannii is the most notorious member of the genus (1), primarily due to its high resistance to antibiotics (2–4). A. baumannii is a Gram-negative coccobacillus opportunist pathogen that mainly causes infections in the respiratory tract, the urinary tract, and the bloodstream and can infect wounds and burns (1, 5). These infections can be challenging to treat since a large proportion of hospital-acquired infections are highly resistant to many antibiotics, including the last-resort drugs colistin and carbapenem (6). A. baumannii mostly acquires antibiotic resistance mechanisms by way of transfer of resistance genes on plasmids, transposons, and integrons (7, 8); however, several efflux pumps, particularly those belonging to the resistance-nodulation-division (RND) family, also play a key role (9). A. baumannii is also capable of forming biofilms on abiotic surfaces such as medical equipment and of surviving in highly desiccated environments (10, 11). All these characteristics contribute to the growing concerns regarding A. baumannii infections.

In 2017, the World Health Organization published its first list of priority pathogens to guide research and development of novel antibiotics. Due to its highly resistant nature, carbapenem-resistant A. baumannii, was categorized as one of the top three critical priority pathogens (12). Additionally, the Centers for Disease Control and Prevention reported that carbapenem-resistant Acinetobacter is an urgent threat to the health care system and that it was responsible for an estimated 700 deaths in the United States in 2017 alone (13). It is now apparent that the conventional ways to discover new antibiotics cannot keep up with the rapid adaptations of this pathogen. Thus, investigation of the resistance mechanisms and virulence factors of this bacterium is vital to aid in the design of effective treatments for A. baumannii infections.

Iron acquisition mechanisms are important virulence factors utilized by bacterial pathogens for survival in a host, and A. baumannii is no exception. Iron is an essential nutrient for nearly all life on Earth (14). It is required for producing energy in the electron transport chain. Iron is also essential for DNA repair, DNA replication, and gene expression functions, as well as other metabolic processes such as activation of enzymatic reactions (15–19). Among bacterial species, iron is generally an essential cofactor for enzymes involved in these processes, namely, cytochromes, succinate dehydrogenases, and catalases (20). It is also required for ribonucleotide reductases, such as NrdAB, involved in DNA replication (21). Iron is critical in pathogens such as Salmonella enterica, including in the central regulatory proteins Fur, Fnr, NorR, SoxR, IscR, and NsrR (22). Not surprisingly, iron is a nutrient requirement for A. baumannii. It is therefore necessary that for infection progression, A. baumannii acquires iron from the iron-limited host environment (23).

HOST IMMUNE SYSTEM IRON-CHELATION SYSTEMS

Human hosts have many defense mechanisms to protect from pathogenic organisms. One such defense mechanism, referred to as nutritional immunity, limits the amount of iron available extracellularly, thereby restricting pathogen access to this essential metal (24). Most iron in the human host is bound to hemoglobin in red blood cells for use in respiration. Any additional iron in the ferric form may be found bound to other high-affinity iron-binding proteins such as transferrin, lactoferrin, or ferritin (25, 26). An innate immune system protein, calprotectin, has metal-sequestering functions that allow it to bind many essential nutrient elements, including iron in the ferrous form (27–29). Calprotectin is a key element in the nutritional immune function of the human host (30, 31).

Transferrin has been shown to inhibit growth of A. baumannii in in vitro assays in which bacterial cells were exposed to human transferrin for 1 h, which resulted in the reduction of CFU by 10% (32). This was found to be a result of transferrin-mediated iron starvation in the growth medium. Another study examined the effects of lactoferrin on A. baumannii biofilm formation in strains isolated from wound infections. Of the isolates tested, 75% experienced significant inhibition of biofilm formation after treatment with human or bovine lactoferrin (33). Supplementation of the growth medium with ferric chloride nullified the inhibitory effects of lactoferrin, indicating that the growth inhibition of A. baumannii isolates resulted from iron starvation. A recent study examining the growth of A. baumannii in media containing calprotectin observed an upregulation in genes involved in siderophore biosynthesis and utilization apparent in both transcriptomic and proteomic analysis (34). This response, typical of limiting iron conditions, confirms that calprotectin also induces iron starvation in A. baumannii.

MECHANISMS FOR ACQUIRING AND UTILIZING IRON IN LIMITING CONDITIONS

Erythrocyte lysis and heme uptake.

Although the human host has most of its iron supply bound to heme groups within hemoglobin molecules, many A. baumannii strains can acquire this heme-bound iron. Like many other pathogenic bacteria (35–37), A. baumannii can gain access to iron pools within erythrocytes by utilizing hemolytic proteins (38, 39) (Fig. 1). In A. baumannii, these include genes that have been identified to encode two phospholipase C genes (plc1 and plc2) and various hemolysin-related genes (38). A study published by Fiester et al. (39) demonstrated significant cell lysis and cell membrane damage in horse erythrocytes coincubated with A. baumannii cultured in iron-chelated media but not when grown in iron-rich medium. An upregulation in the plc1 and plc2 genes, correlated with iron limitations, suggests that these phospholipase C genes are involved in the acquisition of iron via the lysis of erythrocytes. The fact that these genes are found in all sequenced A. baumannii genomes but not in nonpathogenic Acinetobacter baylyi ADP1 strains suggests a possible role of plc1 and plc2 gene products in virulence (39).

FIG 1.

Iron acquisition and utilization mechanisms in A. baumannii within a host organism. (A) Phospholipase C produced by A. baumannii lyses host erythrocytes to harvest heme transported into the cell by way of the HphR TonB-dependent outer membrane receptor (42). (B) The enzyme HemO frees iron for use by the cell. (C) The siderophore acinetobactin binds iron with high affinity and uses the BauA TonB-dependent outer membrane receptor that utilizes proton-motive force and an inner membrane ATP-binding cassette (ABC) transporter composed of BauB, BauC, BauD, and BauE to transport iron into the cell (53). (D) The siderophore baumannoferrin binds iron with high affinity and uses the BfnH TonB-dependent outer membrane receptor that utilizes proton-motive force and an inner membrane ABC transporter to transport iron into the cell (53). (E) The siderophore fimsbactin binds iron with high affinity and uses the FbsN TonB-dependent outer membrane receptor that utilizes proton-motive force and the FeoA transporter to transport iron into the cell (53). (F) FeoAB within the cytoplasmic membrane is important in the import of ferrous iron. (G) NfuA is a cytoplasmic Fe-S cluster protein needed for iron utilization intracellularly. (H) Outer membrane vesicles of A. baumannii are enriched with TonB transporters for acquiring iron. IM, inner membrane; OM, outer membrane.

After lysing erythrocytes, A. baumannii captures iron from heme using heme uptake systems (40) (Fig. 1). A genome analysis of A. baumannii clinical isolates found two heme uptake gene clusters (41). Heme uptake cluster 1 encodes a TonB-dependent outer membrane receptor, a periplasmic heme-binding protein, and an inner membrane ATP-binding cassette (ABC) transporter. Heme uptake cluster 2 encodes a TonB-dependent receptor, an extracytoplasmic function (ECF) sigma factor and its anti-sigma factor, and a putative heme oxygenase (HemO). In the process of importing heme, HphA is an important hemophore secreted from the cell with the two functions of binding hemoglobin and scavenging free heme (42). HphA transfers these iron sources to the HphR outer membrane receptor in a two-component receptor system that incorporates hemoglobin bound iron into A. baumannii cells. This heme uptake system is required for full virulence of A. baumannii (42).

Heme uptake and utilization has been found to be dependent on the abHemO heme oxygenase (41). HemO has been shown to catalyze the oxidative cleavage of heme to biliverdin and CO to release iron in other organisms (43). One study demonstrated that A. baumannii LAC-4, a hyperresistant and hypervirulent strain, can efficiently utilize iron bound to heme in contrast to A. baumannii ATCC 17978 that encodes fimsbactin siderophore gene clusters in the place of hemO (41). LAC-4 was able to grow with heme as the sole iron source while expressing the AbHemO protein, as detected by Western blotting. ATCC 17978 grown with heme as the sole iron source had growth curves similar to those seen when experiencing low-iron conditions, indicating its inability to utilize the extracellular heme (41).

The same study also investigated the substrates that HemO can convert to biliverdin. A. baumannii cells were grown with extracellular heme as their sole iron source. Extracellular heme could be differentiated from intracellular heme using isotopic labeling of carbon, and the composition of intracellular versus extracellular heme in the medium was quantified. The results were consistent with extracellular heme uptake and metabolism by HemO, and any intracellular heme found in the medium was thought to be a result of heme turnover due to maintenance of iron homeostasis (41).

Siderophores.

Siderophores are molecules that are designed to be secreted in the iron acquisition process. They provide advantages as they allow for extracellular scavenging of iron (44, 45). Siderophores bound to iron typically bind a receptor and are allowed entry into the bacterial cell (Fig. 1) (46–48). They are further processed to remove the iron, which is then directed to essential physiological processes as required (49). Three classes of siderophore iron chelators have been identified in A. baumannii. These classes include acinetobactins, fimsbactins, and baumannoferrins (Fig. 1) (50–52). All three siderophore gene clusters are significantly upregulated in iron-limiting conditions, and addition of iron to the media causes repression of siderophore production. Zinc depletion results in gene expression increases of 10- to 1,000-fold less than that induced by iron depletion; however, even this modest expression change may suggest that although siderophores are mainly iron-regulated, they can be partially regulated by zinc under conditions of prolonged metal constraints (53). It is thought that the various siderophores have some redundancy due to research demonstrating that deletion of one siderophore system in strains with multiple classes does not drastically affect overall siderophore activity. In fact, expression of just one of these siderophore systems is enough to provide cells with sufficient iron-chelating abilities in vitro (53).

The acinetobactin class of siderophores is highly conserved in clinical isolates of A. baumannii. Acinetobactin is a versatile metal chelator that has two isomers: one that is most active at neutral and basic pH and another that is more active at acidic pH (54, 55) Transcriptional profiling of the acinetobactin gene cluster found that all genes in this cluster are significantly upregulated in low-iron conditions (56). The acinetobactin siderophore was shown to be important in the acquisition of iron from host serum proteins such as transferrin and lactoferrin (53). Mutants lacking acinetobactin grown in metal-chelated minimal medium with human serum were seen to have impaired growth compared to the fimsbactin and baumannoferrin mutants when grown under identical conditions. The reduced growth observed was similar to that seen when the mutants were grown with human transferrin as the sole iron source (53). The importance of acinetobactin in the pathogenesis of A. baumannii was also shown in a mouse infection model, in which it was the only siderophore found to be essential for pathogenesis (53). In fact, A. baumannii ATCC 19606 expresses acinetobactin as its sole siderophore system and is still able to survive in the iron-limiting conditions within invertebrate and vertebrate hosts, (57, 58), underscoring its importance in the virulence of A. baumannii.

One study using human alveolar epithelial cells demonstrated that acinetobactin is needed for intracellular A. baumannii infection, as acinetobactin mutant strains had significantly less intracellular persistence compared to the wild-type ATCC 19606 (59). These same strains were again used to infect alveolar epithelial cells to monitor rates of human cell apoptosis. Acinetobactin mutants had a 2-fold decrease in apoptosis rates, and acinetobactin receptor mutants had a 24-fold decrease in apoptosis rates compared to the wild type. This demonstrates that A. baumannii expressing the acinetobactin siderophore system cause more cell damage (59). These infection assays also confirmed that the acinetobactin system is required for A. baumannii’s virulence in Galleria mellonella and mouse models (59).

Acinetobactin may also be responsible for inhibiting some commensal bacteria. When coplating A. baumannii ATCC 17978 with common skin and nose commensals in iron-poor conditions, Knauf et al. (60) found that growth of all Staphylococcus species, as well as Corynebacterium striatum, were significantly inhibited. With the help of a transposon mutant library using A. baumannii AB5075, the acinetobactin biosynthesis and transport genes were identified as being a large proportion of the genes involved in inhibition of Staphylococcus epidermidis library (60). Acinetobactin mutants also displayed reduced inhibition of Staphylococcus hominis and C. striatum compared to the wild-type A. baumannii. Although other siderophores may be involved in commensal inhibition by way of iron competition, acinetobactin is thought to be the main siderophore responsible for this observed effect (60).

Baumannoferrin was first discovered while studying A. baumannii AYE. This strain could not produce acinetobactin due to a mutation in entA. The entA gene is located outside the acinetobactin gene cluster and is important in the biosynthesis of 2,3-dihydroxybenzoic acid, a precursor to acinetobactin. A. baumannii AYE was, however, still able to survive in low-iron conditions despite the lack of ability to produce acinetobactin (58). Further examination of clinical isolates of A. baumannii found three gene clusters coding for siderophores. One of these gene clusters was identified as acinetobactin, and another was found to code for a hydroxamate siderophore found to be conserved in the isolates examined, including A. baumannii AYE (38). This putative gene cluster, now known as baumannoferrin, was thought to be involved in iron acquisition when it was determined that components of this cluster were upregulated in low-iron conditions (56). Characterization of baumannoferrin found that this iron chelator is composed of citrate, 1,3-diaminopropane, 2,4-diaminobutyrate, decenoic acid, and α-ketoglutarate. Baumannoferrin was found to have two isomers, baumannoferrin A and baumannoferrin B, that differ by only one double bond. The gene cluster encoding baumannoferrin contains genes needed for its biosynthesis, transport, and intracellular utilization (58). Baumannoferrin is thought to be the only siderophore used by A. baumannii AYE, proving that it is sufficient alone for survival in iron-deficient environments (58). A. baumannii AYE’s virulence in G. mellonella is comparable to other A. baumannii clinical isolates, demonstrating that A. baumannii AYE is not dependent on acinetobactin for pathogenesis. This contrasts with A. baumannii ATCC 19606, which encodes all three siderophores but still requires a functioning acinetobactin system for virulence in G. mellonella and mouse models (53, 58).

Fimsbactins are a third class of siderophore found in less than 10% of sequenced A. baumannii strains (61). Some of these fimsbactin-containing strains include A. baumannii ATCC 17978 and A. baumannii 6013150 (62). Fimsbactins are composed of two catecholates and one hydroxamate, and they have a backbone of l-serine/threonine and putrescine used as iron-chelating motifs (50). Fimsbactin A is the primary siderophore, while fimsbactins B through F are thought to be biosynthetic intermediates (61). A study looking to prove the siderophore characteristics of fimsbactin grew A. baumannii ATCC 17978 in medium with low-iron conditions that allowed for only weak growth. It was seen that both fimsbactin alone and fimsbactin preloaded with ferric iron improved cell growth in time-dependent and dose-dependent manners (61). However, interestingly, this same study observed that growth enhancements caused by addition of the exogenous acinetobactin siderophore to the medium were antagonized by the addition of the fimsbactin A. It is hypothesized that the observed antagonistic effect may be due to pathway competition (61). Both fimsbactin and acinetobactin are synthesized using the nonribosomal peptide synthetase (NRPS) assembly systems. They share the 2,3-dihydroxybenzoic acid (DHBA) and l-threonine precursors (51, 62). It was also found that these two siderophores directly compete for binding to the BauB periplasmic siderophore-binding protein (61). Performing an analysis of the acinetobactin and fimsbactin gene clusters, Conde-Pérez et al. (52) found that all acinetobactin genes except for entA have a potential redundant equivalent in the fimsbactin gene cluster. This group hypothesized that fimsbactin may therefore act as a replacement in the case that acinetobactin is inactivated, possibly explaining why fimsbactin is rarer among A. baumannii isolates (52). However, the effects of the redundancies between acinetobactin and fimsbactin, as well the potential relationship of baumannoferrin with these two siderophores, need to be investigated further.

Transport of siderophore-bound or heme-bound ferric iron across the outer membrane and into the periplasmic space is an active process, energy for which is provided by the TonB-ExbB-ExbD protein complex via proton-motive force (Fig. 1) (63, 64). TonB associates with ExbB and ExbD by way of a short transmembrane N-terminal domain (65). A proline-rich spacer within the TonB structure is required for formation of a complex between TonB and the outer membrane protein FhuA in vitro (66, 67). Removal of this spacer, however, does not affect TonB function in vivo (68, 69). Further research is needed to investigate the existence of this outer membrane protein and TonB complex in vivo.

Approximately 21 putative TonB-dependent outer membrane transporter genes, some of which are iron-regulated, have been recognized in A. baumannii genomes (70, 71). In the case of the TonB, three coding genes have been identified and named tonB1, tonB2, and tonB3 (72). The iron-regulated tonB3, overexpressed in iron-chelated compared to iron-rich conditions (56), has been shown to be necessary for survival of A. baumannii in iron-limiting conditions and is also essential for A. baumannii virulence in G. mellonella insect models and mouse mammalian models (56, 73). Additionally, tonB1 tonB2 double mutants in A. baumannii ATCC 19606 have significantly lower virulence than the parental strain, but inactivation of tonB1 or tonB2 alone does not significantly affect virulence (72). These findings together suggest that tonB3 plays a major role in transport of iron chelators, while tonB1 and tonB2 play more minor roles in this process (72).

The tonB3 gene of the TonB complex has a Fur-controlled promoter, making it iron regulated. Fur-controlled promoters have transcription blocked when iron levels are sufficient, due to the binding of ferrous iron to Fur creating a repressor complex. Transcriptional activation occurs during iron starvation by the binding of apo-Fur to the promoter (74).

TonB transporters have also been found to be enriched in outer membrane vesicles (Fig. 1) (75). Outer membrane vesicles (OMVs) are produced by all Gram-negative bacteria and are capable of carrying iron and other nutrients (75, 76). In a study of A. baumannii DS002, the OMVs were found to contain 19 different TonB transporters. In silico analysis showed that seven of these transporters contained structural features present in transporters known to be involved in translocation of siderophore-bound iron (75). This suggests that OMVs are involved in capturing siderophores from nearby bacteria in order to transport ferric iron to A. baumannii (75).

FeoAB ferrous transport system.

Feo is the main system for ferrous iron transport in Gram-negative bacteria (Fig. 1) (77). The FeoAB import system is conserved in A. baumannii strains along with its regulator FeoC (70). The feo operon encodes FeoA, a cytosolic protein with unknown function; FeoB, the protein responsible for active transport of ferrous iron across the cytoplasmic membrane; and FeoC, a cytosolic transcriptional repressor (78). Interestingly, feoB deletion does not diminish A. baumannii growth in iron-poor M9 minimal medium and does not prevent siderophore production or lessen A. baumannii virulence. It is hypothesized that ferric iron uptake is more active than ferrous iron transport under these conditions (73). The Feo system does, however, seem to be necessary for growth of A. baumannii in human serum containing iron chelated to transferrin. Mutants with feoB deletion had a 4-fold reduction in cell density compared to the wild type when grown in human serum, suggesting that the Feo system may assist in bacterial proliferation in A. baumannii (73). A. baumannii feoB mutants experienced faster and died in considerably greater numbers when exposed to the complement systems within normal human serum than did the wild-type cells (73). The somewhat contradictory findings that FeoB is not required for growth in iron-poor minimal media but is needed for growth in iron-chelated human serum need to be investigated further. Additionally, although that FeoB was not found to be essential for A. baumannii growth in minimal medium, it was surprisingly observed (through RT-quantitative PCR [qPCR]) that feoB was significantly upregulated in iron-poor conditions (compared to iron-supplemented conditions) (73). Why feoB’s expression is upregulated under these conditions when its deletion does not impact A. baumannii’s growth remains to be explored further.

The feo operon, similar to tonB3, is iron regulated as putative ferric uptake regulator (Fur) boxes were identified within the promoters of these genes (73). The Fur box for the Feo system is found downstream of the transcriptional start site in feoA promoter (73), thus further indicating that the expression of the Feo system responds to iron concentrations.

NfuA Fe-S cluster protein.

With the goal of studying genes involved in iron acquisition, a study screening an A. baumannii ATCC 19606 transposon mutant library identified a mutant with the gene nfuA disrupted that could not grow in iron-chelated conditions. NfuA is structurally similar to orthologous proteins in Escherichia coli and Acinetobacter vinelandii that are Fe-S cluster proteins (79). It was therefore hypothesized that nfuA encoded an Fe-S cluster protein in A. baumannii and that these cytoplasmic proteins important in Fe-S cluster formation were probably involved in iron utilization intracellularly, as Fe-S clusters are known to make up a major part of the iron within the cell (Fig. 1) (79, 80). RT-qPCR analysis of nfuA expression found that transcripts of this gene were upregulated (~2-fold) in iron-chelated conditions compared to iron-rich conditions (79). Purified NfuA was shown to bind to iron, which may support the hypothesis that the NfuA is an important intracellular iron source in A. baumannii in iron-limiting conditions. Western blot experiments showed comparable amounts of BauA, a component of the acinetobactin outer membrane receptor, in the mutant and wild-type strains, demonstrating that the Fe-S cluster mutant strain still had a functioning siderophore system (57, 79). This indicates that the lack of growth of nfuA mutants in iron-chelated environments is likely due to a deficiency in intracellular iron metabolism (79). The growth reduction effects noticed in nfuA mutants could be alleviated by adding inorganic iron to the media or by complementing the mutants with either the nfuA gene from the parental strain or the nfuA ortholog found in E. coli. The nfuA mutants were also found to have a significantly decreased capacity to kill G. mellonella, demonstrating that this gene is involved in A. baumannii virulence (79). Further studies are required to better understand NfuA’s contribution to intracellular iron metabolism in A. baumannii.

RELATIONSHIP BETWEEN IRON ACQUISITION AND OTHER VIRULENCE FACTORS

A. baumannii is infamous for causing life-threatening hospital-acquired infections. This bacterium’s ability to infect immunocompromised individuals is enhanced by virulence factors that allow A. baumannii to easily colonize hospital surfaces, including the surfaces of catheters and ventilators (1, 81). Virulence phenotypes that assist A. baumannii in persisting on biotic and abiotic surfaces include its ability to adhere to surfaces, resistance to desiccation, and ability to form biofilms. Additionally, increased multidrug resistance is another factor allowing A. baumannii strains to survive in hospital environments (81). In the following sections, we discuss how iron acquisition mechanisms influence the virulence of A. baumannii.

Relationship between iron acquisition and cell adherence.

Adhesion to host cells is an important first step in A. baumannii’s pathogenesis. A study investigating the role of the acinetobactin siderophore system in the initial interaction of A. baumannii with A549 human respiratory epithelial cells used ATCC 19606-derived deletion mutants of acinetobactin biosynthesis or acinetobactin receptor genes (59, 82). The absence of acinetobactin biosynthesis or receptor genes did not affect A. baumannii’s ability to attach to the respiratory cells. There was also no difference in the intracellular numbers of A. baumannii within the A549 cells. This indicates that the acinetobactin iron acquisition system may not play a role in initial cell adherence of A. baumannii ATCC 19606 to A549 human respiratory epithelial cells (59).

Recent work, however, suggests that adherence of A. baumannii to host cells involves binding to the host extracellular matrix protein, fibronectin (83, 84). One of the outer membrane proteins that A. baumannii uses to bind to fibronectin has been identified as a TonB-dependent copper receptor protein (83). This protein’s characterization led to the study of the relationship between the TonB system and the adherence of A. baumannii to fibronectin. Using fibronectin-coated polystyrene plates, it was observed that the attachment of the tonB1 deletion mutant was not different from that of the wild-type strain. However, the tonB2 deletion and tonB1 tonB2 double deletion mutants displayed a reduction in the ability of A. baumannii to adhere to fibronectin. Despite the fact that tonB1 and tonB2 are not essential and in addition to a role in siderophore-mediated iron acquisition, they have a greater role in assisting A. baumannii in binding to fibronectin than TonB1 does (72).

In vitro findings above were also confirmed using A549 human respiratory epithelial cells. Monolayers of A549 cells were each infected with A. baumannii deletion mutants (listed above) (72). For ATCC 19606, 10% of the bacterial cells inoculated adhered to the A549 monolayer. Similar to the in vitro experiments, adherence of tonB1 deletion mutant was not significantly different from that of the wild-type strain. However, the tonB2 deletion mutant and tonB1 tonB2 double deletion mutant displayed a significant reduction in adherence to the A549 cells compared to the wild-type strain. Since TonB is present in the inner membrane spanning the periplasmic space, it is not likely to interact with fibronectin directly and may also help explain its minor role in iron acquisition. It is therefore suggested that TonB2 perhaps mediates the export of fibronectin-binding protein(s) to the outer membrane (72). Nevertheless, taken together, it can be concluded that TonB2 plays a prominent role in A. baumannii ATCC 19606’s adherence to a fibronectin-coated abiotic surface or fibronectin present in A549 human respiratory epithelial cells.

Relationship between iron acquisition and cell motility.

A. baumannii does not have flagella but is capable of twitching motility on semisolid agar (56, 85–89). Melissa Brown’s group at Flinders University carried out a transcriptomic analysis under iron-limiting conditions to identify iron acquisition systems in A. baumannii ATCC 17978 (56). Their data showed that 18% of motility-related genes in A. baumannii were significantly downregulated in reduced-iron conditions. Many of these downregulated genes belonged to the type I and type IV pilus systems (56).

Type I pili are characterized by the CsuA, CsuB, and CsuE proteins and are predicted to form part of the pilus structure in A. baumannii, while CsuC is a chaperone protein that helps to fold the pilus rod subunits. CsuD is an outer membrane protein for the assembly and extension of the type I pilus (90, 91). Expression of csuC, csuE, and csuB was downregulated under reduced-iron conditions (56). Type IV pili were initially found to be involved in twitching motility in Pseudomonas aeruginosa and have been characterized in A. baumannii as well. Genes involved in the biosynthesis of type IV pili such as pilB, pilC, pilD, pilT, pilU, comL, comM, comN, comO, and comQ were significantly downregulated in iron-starvation conditions, and so were chemosensory and regulatory genes related to type IV pili, such as pilG, pilH, pilI, pilJ, pilR, pilS, and chpA (56). These results were confirmed using motility assay, as A. baumannii ATCC 17978 was incapable of migrating on the surface of on iron-chelated semisolid agar. It is speculated that since motility has high energy requirements in bacteria, the inability to migrate may be a stress response in A. baumannii within iron-limiting environments (56).

Relationship between iron acquisition and biofilm formation.

The persistence of A. baumannii in abiotic environments is dependent upon its ability to form biofilms (92). Biofilms, formed typically under harsh environmental conditions, are composed of bacterial cells, extracellular polymatrix, and extracellular DNA that can adhere to biotic and abiotic surfaces (93–96). One study in A. baumannii clinical strains observed that as iron concentration in the growth medium decreased, the overall siderophore activity increased as expected (97). However, interestingly the activity of acyl homoserine lactones (AHLs), autoinducer signals important in biofilm formation in A. baumannii, also increased as the iron concentration decreased (97). Finally, biofilm formation was stronger in A. baumannii strains when iron concentrations were low and weaker when iron concentrations were high. Taken together, these observations indicate that iron plays a regulatory role in AHL production, which affects biofilm formation (97). However, molecular mechanisms that regulate AHL production in A. baumannii in response to the iron concentrations remain largely unknown.

In addition to being involved in antibiotic resistance, there is increasing evidence to suggest that efflux pumps are involved in biofilm formation (98). The observation that the tripartite efflux pump MacAB-TolC is overexpressed in mature biofilms of A. baumannii ATCC 17978 led to the investigation of the proteome of macAB-tolC deletion mutants (99). Proteomic analysis showed a 2-fold decrease in the expression of the negative Fur regulator in the macAB-tolC deletion mutant. The Fur regulator is known to inhibit synthesis of siderophores when iron levels are sufficient (99, 100). Expression of BauA, involved in acinetobactin import, increased 10-fold in the macAB-tolC deletion mutant. The BfnH and TonB-dependent receptor proteins involved in baumannoferrin transport were also overexpressed. Interestingly, expression of many proteins involved in acinetobactin and baumannoferrin biosynthesis were downregulated in macAB-tolC mutants. These proteins include BfnL involved in baumannoferrin production and BasB, BasE, and BasF proteins for acinetobactin synthesis. BauB (involved in the import of acinetobactin) and BarB (involved in the export of acinetobactin) were also downregulated. There was also a decrease in the production of siderophores in the macAB-tolC deletion mutant (99).

In macAB-tolC mutants, five proteins involved in the phenylacetate (PAA) catabolic pathway were downregulated up to 15-fold (99). The alteration in iron homeostasis in macAB-tolC mutants likely affects synthesis and expression of the PAA operon (99, 101). The PAA pathway produces succinyl-CoA and acetyl-CoA by the degradation of aromatic compounds and is determined to be involved in A. baumannii virulence (102, 103). This discovery was made when Cerqueira et al. (103) used two A. baumannii deletion mutants of genes encoding proteins needed early on in the PAA pathway (paaA and paaE). Testing these mutants in mouse septicemia models revealed that paaE mutants are significantly attenuated (103). Additionally, it was recently determined that A. baumannii paaB mutants with excess PAA (104, 105) are sensitive to oxidative stress and have increased antibiotic susceptibility, establishing the relationship between the PAA pathway and A. baumannii virulence (106). Pérez-Varela et al. did not discover virulence attenuation in mutant strains using a Caenorhabditis elegans survival assay (107). There is, however, a link between PAA and another nutrient essential mineral, manganese. In fact, a manganese-responsive transcriptional regulator in ATCC 17978, MumR, is responsible for virulence, as well as activation of the manganese transporter MumT, during oxidative stress, alleviating such oxidative effects (108). It is thought that PAA metabolic intermediates may act as a reservoir of manganese, which then acts as a cofactor for enzymatic detoxification of reactive oxygen species. In fact, in the case of Borellia burgdorferi, use of iron has all but ceased and been replaced with manganese (109). This substitution of manganese for iron has also been observed in E. coli via an alternate ribonucleotide reductase with an affinity for both iron and manganese and is active under iron-limiting conditions (21). Additional research into the role that iron plays in regard to the PAA pathway and if a comparable pool of iron is generated via metabolic intermediates is an interesting avenue for investigation. Considering catalases require iron for detoxification of hydrogen peroxide, an on-demand supply of iron must be made available, possibly in a manner similar to that of manganese. Further research will be also needed to better understand the relationship between the MacAB-TolC efflux pump’s biofilm forming functions and its potential role in iron homeostasis, as well as the control of the PAA virulence pathway.

Relationship between iron acquisition and antibiotic susceptibility.

Multidrug resistance of A. baumannii warrants finding effective treatment options for such infections. Harnessing the knowledge of iron uptake and infection treatment, a novel antibiotic called cefiderocol was approved for clinical use in 2019 by the U.S. Food and Drug Administration (FDA). It is a conjugated molecule joining a cephalosporin with a catechol moiety (110, 111). This enhances entry of β-lactam antibiotic into the periplasmic space of the cell via the iron transport system (49, 112). After entry, the iron molecule dissociates from the siderophore and crosses the inner cell wall membrane to enter the cytoplasm (111, 113). The pharmacologically active component remains in the periplasm, where it exerts its effects by binding penicillin-binding proteins to inhibit the synthesis of peptidoglycan (111). Cefiderocol has been found to be active against a number of Gram-negative bacilli, including multidrug-resistant (MDR) Klebsiella pneumoniae, P. aeruginosa, Stenotrophomonas maltophilia, and A. baumannii (110, 114). Several factors make it difficult for bacterial cells to develop resistance to cefiderocol. Utilizing iron transport as a Trojan Horse strategy for antibiotic entry allows high concentrations of antibiotic to reach the site of action, avoiding potential porin-mediated resistance. Further, cefiderocol avoids β-lactamase hydrolysis and is not a good substrate for efflux pumps, making resistance to this antibiotic difficult to acquire (112, 115).

Most of the work on the mechanism of action cefiderocol has been done in P. aeruginosa PAO1 with the PiuA ferric iron transport system implicated in uptake of this antibiotic (112); however, no homologous sequences to piuA were found in A. baumannii, and therefore cefiderocol is likely to enter A. baumannii via a novel mechanism. A recent study showed that the expression of a TonB-dependent receptor, PirA, was required for the efficacy of cefiderocol against A. baumannii (116). However, further studies are needed to ascertain PirA’s role in cefiderocol activity in A. baumannii; in addition, the role of other TonB-dependent receptors cannot be ruled out (116).

Not surprisingly, some instances of resistance to cefiderocol have been reported (117, 118). These include mutations in genes for siderophore synthesis and regulation, iron uptake, two-component regulatory systems, and penicillin-binding proteins (111, 119). MIC₉₀ for A. baumannii has been reported to be 2 μg/mL, classified as within the susceptible range for this antibiotic (117, 119). However, when limited to MDR isolates of A. baumannii, the MIC₉₀ was 4-fold higher at 8 μg/mL, falling into the intermediate clinical breakpoint category (114, 117, 119, 120). Thus, MDR isolates are more likely to develop resistance than those in the susceptible category. Some MDR isolates had MICs as high as 256 μg/mL (117, 119, 120). Therefore, although this novel antibiotic, designed to take advantage of iron uptake systems, may be effective for many Gram-negative bacilli, including some A. baumannii strains, most MDR A. baumannii strains still seem to have remarkably low susceptibility to cefiderocol. Recent evidence suggests that inconsistencies in cefiderocol efficacy in clinical trials may result from cefiderocol herteroresistance, especially in carbapenem-resistant A. baumannii infections (121). Further characterization of resistant isolates will be important to understand the mechanisms of cefiderocol resistance in A. baumannii. Nonetheless, cefiderocol highlights the potential of exploiting the iron acquisition systems for designing therapeutic options against pathogens.

CONCLUSIONS

The versatility of iron-acquisition and utilization pathways in A. baumannii plays an important role in its ability to cause infections. Its ability to infect a host to a large part is dependent on its capacity to acquire iron from the environment. Iron concentrations have been shown to influence cell motility via the modulation of type I and type IV pili systems. Biofilm formation by A. baumannii, an important virulence factor, is also affected by the amount of iron present in the environment. At a molecular mechanistic level, there remain questions regarding iron acquisition and the role of this micronutrient in the fitness of A. baumannii. The exact relationship between iron levels, AHL production, and biofilm formation remains to be discovered. Further investigation into the function of the minor iron acquisition systems, namely, TonB1 and TonB2, is still required, as their exact role in fibrinogen adherence is unknown. The relationship between feoB and iron acquisition in iron-chelated human serum is not well understood but leaves avenues for future exploration. The link between macAB-tolC, siderophore production, and PAA metabolism is incredibly interesting and deserves further research, as the PAA pathway has been hypothesized to provide an intracellular bioavailable source of manganese in response to oxidative stress. The link between iron homeostasis and PAA metabolism remains to be studied in great detail. Finally, the receptor for cefiderocol needs to be determined. If the transport system can be identified, then cefiderocol heteroresistance in carbapenem-resistant A. baumannii can be addressed, and treatment failures can be avoided. Further advances in the field of iron acquisition mechanisms used by A. baumannii will contribute to the knowledge of host-pathogen interactions, and a better understanding of iron acquisition and utilization pathways is certainly going to help efforts directed toward designing effective treatments against A. baumannii.

Biographies

Shoshana Cook-Libin is a B.Sc. student in the Department of Microbiology at the University of Manitoba. She spent two semesters in Ayush Kumar’s laboratory studying efflux-mediated antibiotic resistance in Acinetobacter baumannii. She will be joining the School of Medicine at the Rady Faculty of Health Sciences at the University of Manitoba in the fall of 2022.

Ellen M. E. Sykes is a Ph.D. candidate in the Department of Microbiology at the University of Manitoba. Prior to joining Ayush Kumar’s laboratory at the University of Manitoba, she received her B.S. from University of Alberta, Edmonton. Currently, she is studying the environmental isolates of Acinetobacter baumannii to understand genetic elements that make this organism a problematic human pathogen.

Vanessa Kornelsen is a Ph.D. candidate in the Department of Microbiology at the University of Manitoba. She completed her B.S. in microbiology from the University of Manitoba. Her current research interests include understanding the physiological role(s) of antibiotic efflux pumps in Acinetobacter baumannii.

Ayush Kumar Ph.D., is a professor in the Department of Microbiology at the University of Manitoba. He was the President of the Canadian Society of Microbiologists in 2020 to 2021. His laboratory studies efflux-mediated antibiotic resistance mechanisms in Gram-negative bacterial species, Acinetobacter baumannii and Pseudomonas aeruginosa. He is particularly interested in the studying the mechanisms that control the expression of efflux pumps in these organisms and using the knowledge to gain insights on the physiological functions of efflux pumps. He also studies the microbiological quality of drinking water from First Nations communities in Manitoba. In addition, he is interested in understanding barriers that various marginalized groups face in science.