The Outer Membrane Proteins OmpA, CarO, and OprD of Acinetobacter baumannii Confer a Two-Pronged Defense in Facilitating Its Success as a Potent Human Pathogen

1The Gram-negative coccobacillus Acinetobacter baumannii is an aerobic opportunistic pathogen responsible for some of the most morbid hospital-acquired infections. The global threat from this pathogen comes from its high rate of resistance gene acquisition leading to rapid emergence of multidrug-resistant (MDR) clinical isolates. Increasing number of studies show frequent isolation of carbapenem and colistin-resistant strains of A. baumannii from clinical settings. Swift accumulation and dispersion of antibiotic resistance markers along with the ability to cause urinary tract infections, skin and soft tissue infections and wound infections makes A. baumannii, a pathogen of great significance for both humans and animals. In light of this, the World Health Organization (WHO) categorized this organism as a priority-1 critical pathogen for which discovery of new treatment options is of utmost importance. The potency of A. baumannii as a successful pathogen can be elaborated by the high number of deaths associated with its infection. A recent finding showed that bacteremia caused by multidrug-resistant (MDR) A. baumannii exhibited 56.2% mortality rate among infected patients.

2A plethora of virulence factors are elucidated by A. baumannii, some of which include, but are not limited to, porin proteins, efflux pumps, outer membrane vesicles, metal acquisition systems, secretion systems, phospholipases, and capsular polysaccharides. Recent advances in research have not only provided better knowledge of these determinants, but also shed light on how these can be used as potential drug targets. However, growth conditions like temperature, oxygen content, osmolarity, and media components regulate the expression of porins in A. baumannii. Transcriptional and post-transcriptional regulatory networks also determine the virulence and antibiotic resistance of A. baumannii.

3Among the vast diversity of antibiotic resistance and virulence determinants and A. baumannii specific regulatory networks, one group of bacterial proteins, termed outer membrane proteins (OMPs) due to their localization, have been studied with utmost interest due to their distribution, functional relevance and stipulated role in both antibiotic resistance and virulence. OMPs in general are beta barrel-shaped monomeric or trimeric porins (Table 1) that allow diffusion of small molecules into and out of periplasmic space of Gram-negative bacteria. A. baumannii outer membrane holds scores of OMPs including OmpA, CarO, OprD- like OMPs, Omp 33-36 kDa, AbuO, TolB, DcaP, Oma87/BamA, NmRmpM, CadF, OprF, etc.. OMPs participate in a wide range of functions that assist the bacterium in enduring the harsh environmental conditions, in combating the threat posed by antimicrobial compounds, host, and surprisingly, in degrading crude oil. Immunization with A. baumannii OMPs ensued significant rise in protective immune parameters and antibodies against OMPs passively protected experimental animals. Clinical studies frequently identify differential expression of OMPs in antibiotic resistant A. baumannii strains, establishing their role in conferring resistance. Here, we explore and summarize how antibiotic resistance and virulence in A. baumannii is mediated by different OMPs like OmpA, CarO and OprD.

4OmpA, a beta barrel-shaped monomeric protein is one of the most abundant OMPs, which has been reported to impart drug resistance to A. baumannii by allowing slower diffusion of negatively charged beta-lactam antibiotics and virulence by its toxicity to host cells. Clinical isolates of A. baumannii overexpressing OmpA arbitrate higher morbidity and even mortality in patients. The global repressor H-NS binds to the promoter region of OmpA gene and gene locus A1S_0316 and the two component system BfmSR function as a possible anti-repressor and repressor of OmpA in A. baumannii, respectively.

5Being the most abundant porin in A. baumannii, the role of OmpA in antibiotic resistance was more prominent in disruption mutants of the gene, which showed increased susceptibility to nalidixic acid, chloramphenicol, aztreonam, imipenem, and meropenem. Besides diffusion, research indicates that OmpA possibly couples with efflux pumps and forces out antibacterial compounds from the periplasm. OmpA also couples to A. baumannii peptidoglycan (PG) via its C-terminal region, where Asp271 and Arg286 bind to diaminopimelic acid of PG. This binding may regulate outer membrane vesicle (OMV) production and the membrane stability in the bacteria. OMVs with OmpA in their membrane mediate antibiotic resistance by actively siphoning extracellular drugs. Recently, resistance to colistin, a last-resort antibiotic, was also attributed to the presence of OmpA in A. baumannii. An isogenic mutant of OmpA resulted in loss of cell wall integrity, thus making the bacterium 20-fold more sensitive to colistin and 5.3 fold more sensitive to trimethoprim than wild type A. baumannii. The distinctive role of OmpA in conferring antibiotic resistance thrusts researchers to discover novel antibacterials against the protein. In one study, a novel diazabicyclooctenone beta-lactamase inhibitor that inhibits major classes of carbapenemases and in turn potentiates sulbactam activity was shown to be OmpA-dependent. OmpA blockers can function synergistically with last resort antibiotics like colistin in eradicating MDR strains of A. baumannii. OmpA also interacts with antimicrobial peptides (AMPs) of diverse origin and confers resistance against them. Minimum inhibitory concentrations of human AMP LL-37 and bovine AMP BMAP-28 increased upon binding to N-terminal region of OmpA. The multifaceted role of OmpA in A. baumannii membrane permeability and cell wall integrity indicates its potential as a candidate for novel antibacterial development via chemical genetic screens.

6Besides their distinguishable role in antibiotic resistance, OMPs confer virulence to A. baumannii. The bacterium is capable of invading and persisting in host epithelial and immune cells (Figure 1). The primary requisite for invasion is to adhere to the host cells, which is mediated by many virulence factors expressed by A. baumannii viz., OmpA, BapA, fimbrial like protrusions. A. baumannii adherence to host cells can be both host cell and bacterial sequence-type specific. For instance, two types of adherence patterns have been elucidated in A. baumannii; dispersed adherence of bacteria to the host cells, and adherence of clusters of bacteria at localized areas of the host cells. Bacterial clusters can be a result of amyloidogenic BAP protein mediated biofilm formation. Interestingly, OmpA specifically mediates bacterial binding to healthy cells than cancerous cells. A. baumannii cells devoid of OmpA were found to be less virulent to human airway epithelium due to decreased adherence to cells and formed weaker biofilms.

7Following adherence to host cells, A. baumannii invades into the cell cytoplasm. The bacterial penetration into epithelial cells is microfilament- and microtubule-dependent following zipper-like mechanism. Upon internalization, A. baumannii cells localize to membrane-bound vacuoles and finally traffic to the nucleus. Bacterial cells actively divide and finally kill the host cell to release into the blood stream. In this process, OmpA actively assists bacterial invasion, although by unknown mechanisms. Iron homeostasis is found to be a key factor regulating the survival of A. baumannii in the cytoplasm. Other adherence factors like BapA are not found to mediate invasion of A. baumannii indicating that OmpA specific pathways regulate A. baumannii virulence. The functional dynamics of such a feature for a protein can be emphasized succinctly by looking into immune responses in the host.

8In healthy individuals, A. baumannii cells in the blood stream and airways are actively phagocytosed by circulatory or tissue-resident immune cells like macrophages, neutrophils, dendritic cells (DCs), etc. before leading to fulminant A. baumannii sepsis, although the latter is the case frequently encountered in immunocompromised patients. A. baumannii induces host cell cytotoxicity by targeting mitochondrial system and nuclear localization. In epithelial cells, OmpA induces the surface expression of Toll-like receptor 2 and the production of inducible nitric oxide synthase. Phagocytosed bacteria release several structural and cytoplasmic proteins that induce cytotoxicity directly (e.g., Type VI secretion system effector enzymes, and toxins) or indirectly by activating caspases (e.g., OmpA). In macrophages and epithelial cells, OmpA triggers autophagy, albeit incomplete, by preventing the fusion of autophagosomes with lysosomes, activating MAPK/JNK signaling pathway and enhancing the levels of phosphorylated JNK, p38, ERK and c-Jun. Early response in DCs treated with OmpA includes augmenting the expression of CD40, CD54, CD80, CD86 and MHC-I and II surface markers. The marker expression is accompanied by secretion of Th-1 promoting IL-12. However, upon prolonged exposure, secreted OmpA in DCs targets mitochondria and induces production of reactive oxygen species (ROS) ( and 2010). ROS stimulates early-onset apoptosis and delayed-onset necrosis in DCs, thus impairing T-cell response against A. baumannii. Besides induction of ROS, OmpA in conjunction with carbonic anhydrase IX stimulates DCs to generate potent anti-tumor immune response against renal cell carcinoma.

9All these observations in the host indicate that OmpA might be toxic in nature, unlike other outer membrane proteins. The toxicity of A. baumannii OmpA can be attributed to its unique structural features and vast diversity of alleles with conserved domains. OmpA protein is comprised of two domains; an N-terminal 8-stranded β-barrel domain and a C-terminal periplasmic peptidoglycan binding domain. OmpA possesses a basic amino-acid rich signal termed nuclear localization signal (NLS) ‘KTKEGRAMNRR’ on its C-terminal domain. Karyopherin β family proteins on nuclear pore complex recognize the NLS via its lysine (K) residues and shuttle OmpA to nucleus from cytoplasm. It can thus be speculated that OmpA devoid of NLS can be a non-toxic variant. Studies to reduce the host cell toxicity of recombinant A. baumannii revealed the importance of both N and C-terminal regions and the importance of lysine residues in NLS sequence. A synthetic OmpA with mutations at K320 and K322 to Alanine, replacing “NADEEFWN” sequence with “YKYDFDGVNRGTRGTSEEGTL” and deleting N-terminal signal sequence and “VVQPGQEAAAPAAAQ” at C-terminal resulted in least toxic but highly immunogenic OmpA. In addition to NLS, the N-terminal region of OmpA is bioinformatically predicted to be more immunogenic. The epitopes “QIQDSEHSGKMVAKRQ” at position 100–115 and “HTSFDKLPEGGRAT” at position 125–138 are delineated best by Ellipro software. A peptide at N-terminal region located at 24–50 position, “VTVTPLLLGYTFQDSQHNNGGKDGNLT” alone is immunogenic and elicited similar levels serum antibodies like OmpA. It is clear that OmpA is toxic to host when secreted and when intact, it provides antibiotic resistance to the bacterium. Whether these observations can be implied to the clinical manifestations of A. baumannii is yet to be elucidated.

10Carbapenem susceptibility porin or CarO was first reported in imipenem (IMP) sensitive A. baumannii isolates that acquired resistance upon the loss of a 29 kDa protein. CarO is an 8-stranded beta barrel-shaped outer membrane channel protein that does not have a continuous channel ( and 2011;; ) but mediates influx of beta lactams (selectively imipenem) into A. baumannii. However, contradicting these observations, liposome model system embedded with CarO revealed its ability to transport small amino acids such as glycine and ornithine, but not carbapenem antibiotics. Despite this lonesome tangential observation, the excessive evidence from diverse research groups denotes the role of CarO in antibiotic resistance.

11CarO is classified into two sub-groups; CarOa and CarOb; of which CarOb exhibits a two-fold greater specificity for IMP. However, there has been a recent call to rethink the CarO classification system based on phylogenetic analysis. So far, at least six polymorphic variants of CarO have been reported to co-exist in A. baumannii populations with varied specificities to imipenem, highlighting the importance of the protein. The alterations in CarO gene are posited to be a result of rapid adaptation of A. baumannii to diverse habitats and hosts. Besides gene alterations, conformational changes in primary structure, intra-genic insertion sequences, posttranscriptional and transcriptional regulation dramatically affect CarO function (summarized in Supplementary Table 1). The recent identification that CarO is significantly up-regulated in an Hfq deletion mutant strain of A. baumannii indicate that it is kept under post-transcriptional control by the bacterium to regulate its expression in response to the changing environment. Finally, the occasional isolation of antibiotic resistant strains with a loss of CarO gene signifies the diversity of resistance mechanisms in A. baumannii. In contrast to these studies linking carbapenem resistance to the loss of CarO, there are a few reports of the presence of CarO porin on the OM of carbapenem resistant clinical isolates of A. baumannii. However, this can possibly be explained by the “porin-localized toxin inactivation” model, where carbapenemases like Oxa-23 interact with the periplasmic region of OMPs like CarO or OmpA to act as an efficient selective filter to inactivate incoming antibacterial compounds.

12The clinical relevance of CarO has also been ascertained by many hospital epidemiological studies. These revealed that there is a prevalence of CarO deficiency amongst carbapenem resistant isolates expressing BlaOXA and TEM-1 genes among the hospital isolates of A. baumannii. Various carbapenem resistant clinical isolates demonstrated a disruption in the CarO gene by insertion sequences like ISAba1, ISAba125, ISAba825, ISAba10, ISAba15, and ISAba36. When exposed to a high concentration of monovalent cations, A. baumannii release a variety of OMPs including CarO into the surrounding media and becomes more tolerant to IMP stress. This finding implicated that MICs of antibiotics determined in vitro may not help eradicate A. baumannii infection from within the host system, especially in the case of urinary tract infections where there is the presence of a high concentration of monovalent cations like NaCl and KCl.

13The immunological role of CarO protein in A. baumannii is studied inadequately. showed that clinical A. baumannii isolates expressing higher CarO mRNA levels negatively regulated TNF-α, IL-6 and IL-8 in lung epithelial cells. Recently, CarO has been linked to A. baumannii adhesion and virulence in host cells via inhibition of NF-kβ signaling. However, the significance of this observation is debatable as the strain used in the study is ATCC 19606 where expression of CarO is significantly lesser than that of clinical strains.

14OprD was first identified during outer membrane investigations of carbapenem resistant A. baumannii isolates. It is an orthologous protein to a porin involved in the basic amino acid and imipenem transport in Pseudomonas aeruginosa. Crystallographic studies of a conserved P. aeruginosa OprD revealed a monomeric 18-stranded β-barrel structure characterized by a very narrow pore constriction. The amino acid conservation at structural domains between A. baumannii and P. aeruginosa OprD porins indicate its putative function in A. baumannii. However, sequence and homology analysis of A. baumannii OprD showed that it belongs to P. aeruginosa OprQ, a protein involved in resisting low-iron or magnesium and low oxygen stresses. Recombinant A. baumannii OprD did not conduct antibiotics but partially bound to Fe2+ and Mg2+ cations. An isogenic deletion mutant of A. baumannii OprD did not affect MICs of β-lactams, but in A. baylyi spp., a significant reduction in MIC of imipenem, ertapenem and meropenem is observed. Despite these two heralding reports on lack of relationship between OprD and antibiotic resistance in A. baumannii, single nucleotide polymorphisms and insertional elements in OprD have been frequently identified in MDR A. baumannii signifying its role in resistance. For instance,; and showed SNP clusters in OprD in MDR and tigecycline-resistant A. baumannii, respectively. Downregulation of OprD was observed in MDR and pan drug-resistant A. baumannii clinical strains. Insertion of mobile element ISAba1 upstream to the gene was also associated with increased carbapenem MICs of A. baumannii sequence type 107 strains. OprD was renamed to OccAB1 by, while solving its crystal structure. In their work, Zahn et al., resolved structures of four carboxylate channels OccAB1, 2, 3, and 4 and showed that OccAB1 has the largest channel size with corresponding high rates of small-molecule shuttle, including amino acids, sugars, and antibiotics, contrary to previous observations. The particularly large pore size of OccAB1 facilitates the objective translocation of both positive and negative substrates at low energy cost. On the other hand, OccAB2, OccAB3, and OccAB4 mediate hydroxycinnamate, vanillate, and benzoate transport, respectively. Being induced by limitation of metal ions, it can be presumed that OccAB1 may play a significant role in combating host-induced nutritional immunity and stress survival.

15Besides the above three OMPs, a variety of proteins are identified in the outer membrane of A. baumannii with varied expressions and distinctive roles. One of the most abundant OMPs in A. baumannii is Omp33. Crystal structure of the protein revealed its function as a putative gated channel contributing to low permeability of the outer membrane. Intracellular A. baumannii in the host cell expresses DcaP OMP in abundance. Crystallographic studies on DcaP revealed a trimeric porin structure with affinity to dicarboxylic acids and sulbactam. The most abundant OMP under osmotic stress in A. baumannii is Omp38. Intracellular A. baumannii secretes Omp38, which localizes to the mitochondria stimulating the release of proapoptotic molecules such as cytochrome c and apoptosis-inducing factor. Oxidative stress in A. baumannii induces expression of AbuO, a homolog of Escherichia coli TolC OMP involved in resistance to amikacin, carbenicillin, ceftriaxone, meropenem, streptomycin, and tigecycline, and hospital-based disinfectants like benzalkonium chloride and chlorhexidine. Under iron-limiting conditions, a 76-kDa iron-repressible OMP termed fhuE was overexpressed in A. baumannii to facilitate uptake of xenosiderophores desferricoprogen, rhodotorulic acid and desferrioxamine B. Besides fhuE, two other siderophore (acinetobactin) uptake proteins, BfnH and BauA are also elucidated in the outer membrane of A. baumannii. The translation initiation factor EF-Tu, typically a cytoplasmic protein, is also found to localize in the outer membrane in A. baumannii. Membrane associated EF-Tu binds to DsbA protein in the periplasm and assists in disulfide bonding during protein folding. Externally, EF-Tu binds to fibronectin thus mediating host cell adhesion. Decreased expression of a 33–36 kDa OMP and a 29 kDa is associated with imipenem resistance among A. baumannii. Serodiagnostic studies revealed an antigenic 34.4-kDa OMP specific to sera from A. baumannii infected patients. Upregulation of this protein along with downregulation of CarO and OprD was found to mediate imipenem resistance. The protein along with OmpA and TonB-dependant copper receptor was identified as fibronectin binding protein during infection. In silico exploration into the genome of A. baumannii revealed a nuclease (NucAb), BamA (Oma87), FilF, and TolB in the outer membrane as potential vaccine targets. Immunization with these recombinant proteins protected mice from lethal challenge with A. baumannii (, and;;; ). In another effort toward developing a subunit vaccine against A. baumannii infections, a fusion protein of OmpK and Omp22 was synthesized which provided significantly greater protection against A. baumannii challenge in mice than those immunized with either of the two proteins individually.

16One of the critical gaps in combating A. baumannii is deciphering its overall membrane permeability. Significant progress has been made during the last decade in our understanding of how A. baumannii OMPs mediate antibiotic resistance and virulence in the host cells. Remarkable breakthroughs have also been made in understanding the regulatory mechanisms behind OMP expression and the mechanisms of antibiotic uptake. However, these efforts fall short in many aspects. The knowledge about in vitro or in vivo OMP assembly and folding dynamics in lipid bilayers is scarce. The crystal structure of most studied A. baumannii OMP, OmpA is still elusive, although NLS domain structure has been resolved. Due to its complex structure and hydrophobic loops in its structure, expression and purification of recombinant OmpA presents various hurdles. The solution of crystal structure is decisive in molecular dynamic studies tracking the antibiotic entry and exit through OMPs. Many questions still remain unanswered. How does the beta barrel assembly complex in A. baumannii function? What are the different chaperones mediating OMP folding in A. baumannii? Does the expression of OMPs in A. baumannii depend on transcription factors alone or is it small RNA mediated? Although in small number, concerted efforts are directed toward solving these problems. Crystal structures of CarO1, CarO2, OccAB1 through 4, DcaP, PiuA, Omp33 BauA have been resolved. Understanding the magnitude of posttranscriptional regulation in A. baumannii OMP synthesis is a necessary goal, as this aspect has been overlooked till date. In the next few years, it is likely that A. baumannii OMP compendium will be resolved with novel insights into its structure, diversity, biogenesis, and expression, furthering our efforts in confronting antibiotic resistance and virulence in A. baumannii.

17SU, AS, and RP conceptualized the manuscript and contributed the ideas on the texts. SU and AS wrote the first draft of the manuscript. SU and RP edited the subsequent versions. All authors read and approved the final manuscript.

18The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.