A. baumannii is known for its many intrinsic resistance determinants (present irrespective of an antibiotic exposure) that are often missed due to their low-level of resistance displayed phenotypically. However, upon induction mostly due to an antibiotic exposure these resistance genes are either over-expressed or under-expressed (as in case of porins) contributing to very high resistance. In the present study, tigecycline resistant clinical isolate A. baumannii strain JU0126 were induced in vitro for resistance to eravacycline. Whole-cell transcriptome analysis was performed for both eravacycline induced and non-induced strains of A. baumannii strains. In addition, OMVs were isolated from both strains and their proteomes studied from both eravacycline induced JU0126 strains. The whole-cell transcriptome expression was compared with OMVs proteome in JU0126 strains. To better understand the transcriptome profiles of the clinical isolate, an integrated analysis of the results was done with the transcriptome data from already sequenced eravacycline-susceptible quality control strain A. baumannii ATCC 19606 [25] in a similar experimental protocol to the clinical isolate. The A. baumannii ATCC 19606 strain was used as a reference for the study along with the clinical isolate and the comparative study was focused only between the expression profiles of the un-induced and the laboratory-induced eravacycline resistant phenotypes.
Upregulated DEGs/proteins
Genes pertaining to the family of drug efflux and membrane transport were significantly high in expression among both ATCC 19606 and JU0126 strains. The genes that were upregulated in the eravacycline treated ATCC 19606 strain in comparison with untreated strains included majorly of efflux and transporter families. The multidrug efflux RND transporter permease subunit gene and major facilitator superfamily (MFS) transporter were significantly overexpressed in the ATCC 19606 treated strain. Although specifically, AdeB pump and some membrane proteins were upregulated in the eravacycline treated JU0126 strain. The eravacycline-based antibiotic induction in bacterial strains leading to the upregulation of MDR pumps can be supported through some similar prior works. Abdallah et al. (2015), in their study, showed that the increased MIC values to eravacycline up to 4 μg/mL corresponded to increase in the expression of AdeABC MDR pump. However, the upregulation does not always signify the resistance towards the induced antibiotic, as is the case, that no MDR specific resistance towards eravacycline has been reported in Acinetobacter [26]. The enzyme M1 family peptidase is present in many pathogens and is known to be a key enzyme for the survival in these organisms. It was notable that these enzymes were also upregulated in the antibiotic-treated ATCC 19606 strain, signifying the pressure of survival as induced by the presence of antibiotic. RND efflux pumps are a common mechanism involved in antibiotic resistance among A. baumannii, AdeB efflux pump is one of the upregulated proteins in the antibiotic-induced JU0126 strain. In A. baumannii, AdeABC is the first characterized efflux pump belonging to the RND superfamily. The operon codes for a major facilitator superfamily protein transporter protein AdeA, a multidrug transporter AdeB, and an outer membrane protein (OMP) AdeC. Eravacycline resistance in A. baumannii due to AdeABC efflux has not been reported before; hence, this upregulation can be attributed to the induction due to the antibiotic even though it is not a substrate for the pump. Antibiotic induced adeB efflux pump resistance has a major influence on the resistance status of A. baumannii [27]. The role of similar overexpression of adeB has been noted in some MDR isolates resistant to tigecycline in some of the previous research works [28]. Eravacycline induced overexpression of MacA efflux protein of the MacAB–TolC MDR pump expression was reported in K. pneumoniae emphasizing the role of efflux in eravacycline heteroresistance [29]. The next protein that was upregulated in the present study was an Omp38, which is a major porin protein from A. baumannii. OMPs are crucial proteins for antibiotic diffusion and membrane permeability; deficiency of which leads to increased susceptibility to antibiotic. Studies have shown increased production of OMPs, like OmpA38, CarO, OmpW, in the presence of tetracycline, suggesting that the overexpression relates to overcoming antibiotic stress [18]. A. baumannii is an organism that harbours multiple mechanisms for antibiotic resistance, and β-lactamases are a group that tackles the β-lactam drugs efficiently in these organisms. Class C extended-spectrum β-lactamase ADC-26 was seen upregulated in our study in JU0126 strain. The overexpression of ADC is reported to confer resistance to a range of β-lactam antibiotics making the infections caused by A. baumannii difficult to treat [30]. However, the overexpression in the eravacycline treated JU0126 could be due to a random antibiotic stress response because these β-lactamases does not have substrate specificity for a non-β-lactam drug.
Previous reports have demonstrated that OMVs isolated from antibiotics resistance strains help susceptible strains in transferring antibiotics resistance genes and proteins under antibiotic stress condition [21]. Carbapenem-resistant A. baumannii releases OMVs packed with carbapenem resistance-related genes and could undertake the horizontal transfer to carbapenem-susceptible A. baumannii [31]. In one study, OMVs from E. coli were found to seize antibiotics, such as colistin and degrade the antimicrobial peptides like melittin [32]. Moraxella catarrhalis and Staphylococcus aureus also releases OMVs, which carries β-lactamase helping the bacteria to survive in the presence of β-lactam antibiotics [33].
Downregulated DEGs/proteins
The tetracycline group of antibiotics act by binding to ribosomal subunit 30S thereby blocking the aminoacyl-tRNA to bind to ribosomal acceptor site A; hence, inhibiting the protein synthesis [34]. It was reported by Vrentas et al., that the downregulation of RNA synthesis occurs as a result of protein synthesis inhibition [35]. In the present study, the transfer RNAs were downregulated in both the ATCC 19606 and JU0126 strain, which explains the adaptation of the bacterium to pressure, trying to keep the metabolic process minimal, similar reports on the reduced metabolism due to tigecycline induction was done by Liu et al.[36].
In our study, porin proteins were downregulated in the A. baumannii ATCC 19606 strain. The loss or downregulation of porins is a mechanism of resistance, wherein the bacteria reduce the cell permeability preventing antibiotic entry and decrease the susceptibility [37]. The presence of tetracycline leads to differential expression of porins proteins, either increase or decrease of which decides the permeability of the cell envelope. The downregulation of porins in A. baumannii in this present study corresponds to the previous claims on tetracycline leading to the downregulation of numerous porins in Escherichia coli strains [38].
Subcellular localization of proteins from OMVs
The subcellular localization of the proteins expressed in the OMVs from both the eravacycline treated and untreated control strain was identified using pSORT-B 3.0. Their results give crucial information on the function of the protein, which can be compared with their expression pattern in the present study condition (upregulated or downregulated; antibiotic stress or antibiotic resistance). The pSORT-B categorizes the Gram-negative bacterial proteins into five major sites—the cytoplasm, the inner membrane, the periplasm, the outer membrane and the extracellular space [39].
The localization analysis in the current study was done to visualize the effect of antibiotic stress on the OMVs specifically focusing on their proteins and its functions. In both the ATCC 19606 and JU0126 strains, proteins with functions related to resistance and stress were predominant, like the outer membrane proteins, efflux pumps, β-lactamase associated resistance proteins, stress tolerance proteins and peptidases. It is known that the proteins from OMVs aid the invasiveness of the bacteria, and are enriched with toxins, bioactive and virulence proteins. OMVs are a key for bacterial survival with their role in bacterial self-defence, formation of biofilm, antibiotic resistance and host–immune response modulation [40], [41]. The exposure of cells to environmental contaminants (antibiotics) has potentially evolved bacterial OMVs, either with multidrug efflux pumps capabilities or with ability to catalyse degradation by sequestering antibiotics from the extracellular milieu [42], [43].
DEGs pertaining to virulence factors
The proteins belonging to the type VI secretion systems (T6SSs), which are a new type among the bacterial secretion systems, were increased in their expression ranging from 18-fold to a minimum of 7-fold change in the A. baumannii JU0126 eravacycline treated strain compared with the control. T6SSs are associated with the pathogenicity in bacteria with the experimentally proven role in bacterial virulence [44]. T6SSs does a bacteriophage-like contractile injection of effector proteins puncturing into target cells and when they inject antibacterial toxins to competing for bacterial cells, they become ‘antibacterial’ T6SSs [45], [46]. Some of the other genes with differential expression in JU0126 strain alone were LuxR family transcriptional regulator (a crucial protein involved with quorum sensing) with a 7-fold change in expression. These proteins coordinate the expression of virulence factors, biosynthesis of antibiotics and transfer of plasmids, bioluminescence, and formation of biofilms [47].
The efficient induction of eravacycline resistance was evident with a 5-fold change in expression of the gene that encodes 50S ribosomal protein L2, which is an rRNA binding protein and helps in the interaction of 30S and 50S subunits in order for tRNA binding to happen and; hence, peptide bond formation [48]. This contradicts the action of eravacycline which negates the bacterial protein synthesis by binding to the 30S ribosomal subunit, stopping peptide chains formation [9]. In addition, along with ribosomal proteins, genes for the elongation factor G were increased in their expression by 4-fold change, EF-G has two roles; one, during the translocation and the other, in the ribosome disassembly [49]. Genes coding for the protein involved with cell metabolism, such as the d‐alanyl‐d‐alanine carboxypeptidase, a serine peptidase was 4-fold differentially expressed. These proteins are associated with virulence in Acinetobacter sp. [50] and have been experimentally proven to be essential for intracellular replication in some bacteria [51].
The arginine succinyl transferase A (astA) enzyme [52] gene had an 8-fold increase in the expression of A. baumannii ATCC 19606 treated strain. AstA was found associated with healthcare-associated pathogen A. baumannii strains [53], and it has been attributed to the pathogenesis in other bacterial strains like uropathogenic E. coli (UPEC), and was reported as one of the virulence proteins in E. coli [54].
In both A. baumannii ATCC 19606 and JU0126 strains induced by eravacycline, the genes for type I secretion system and elongation factor TU had a positive log2-fold change, with a 3-fold change in the JU0126 strain. The type I secretion system helps in the secretion of proteins from cytoplasm to the extracellular region. They harbour a specific OMP for their export and one among the best-studied is TolC from E. coli [55]. Elongation factor TU is a GTPase also known to perform moonlighting functions on the surface of human pathogens acting as a multifunctional adhesin [56].
DEGs as resistance determinants
Positive differential expression of many genes encoding resistance proteins was observed in both ATCC 19606 and JU0126 strains induced with eravacycline from the RNA sequence analysis. The efflux pumps and the ribosomal protection are the two main resistance mechanisms in A. baumannii to tetracycline class of drugs. In A. baumannii ATCC 19606, genes for all the major efflux pump family proteins had a positive differential expression, such as MFS, RND, multidrug and toxic compound extrusion (MATE) and ABC transporters. A 9-fold change in the expression of gene that codes for MFS transporter, many of which are involved in the drug efflux of antimicrobials, such as tetracyclines, fosfomycin, colistin and erythromycin [57] noted in the ATCC 19606 strain, whereas the JU0126 strain had a negative log2 change in the expression of this transporter. Tet efflux pumps are among the main types that come under MFS transporters, tetA gene codes for an efflux protein that confers resistance to tetracyclines. The A. baumannii has two pump proteins under MFS category (uses proton exchange for a tetracycline-cation), Tet(A) and Tet(B) [58].
A 7-fold increase in the expression of TetR/AcrR family transcriptional regulator gene was observed in the A. baumannii ATCC 19606, induced with eravacycline while their expression in JU0126 strain showed a negative log2-fold. The TetR family of regulators (TFR) comes under the signal transduction systems with the drug–efflux pump regulation as their functional role. The expression of acrAB efflux pump operon is repressed by the AcrR. TetR is a family of tetracycline transcriptional regulator that has a role in the transcriptional control. In the absence of tetracycline antibiotic, TetR binds to the Tet(A) gene to repress its expression. Tet(A) exports tetracycline from the cell before it can exert the protein synthesis inhibition [59].
The overproduction of RND pumps, such as AdeABC, AdeFGH, and AdeIJK is a major factor contributing to the resistance in Acinetobacter[60]. The gene for AdeB/AdeJ proteins had 3-fold differential expression in both the ATCC 19606 and JU0126 strains. AdeB is the multidrug transporter for the AdeABC tripartite efflux pump that expels out an array of antibiotics, such as aminoglycosides, β-lactams, chloramphenicol, erythromycin, and tetracyclines. This positive differential expression of AdeB can be correlated with the prior studies on which it was reported to be the most prevalent with increased expression among the MDR A. baumannii strains in Zhenjiang, China by Yang et al. [61]. Positive 6-fold differential expression of the multidrug efflux RND transporter permease subunit gene was noted in the ATCC 19606 strain, whereas a negative 3-fold decrease in the expression of the JU0126 strain. The ABC transporter ATP-binding protein gene expression was increased by 6-fold in the eravacycline induced ATCC19606 strain when compared with the uninduced strain; however, the MacB protein subunit was under-expressed with a negative 2-fold change in the same strain. The MacA–MacB–TolC is a three protein efflux system that expels out mainly macrolide class of antibiotics, and their expression may not be influenced in a large way by the eravacycline [62]. MATE family pumps are not much related to resistance towards the tetracycline class of drugs and basically confer resistance towards fluoroquinolones and imipenems [63]. However, there was a 7-fold change in the gene expression of MATE family pumps in the eravacycline induced A. baumannii ATCC 19606, but negative differential expression of negative 5-fold change in the JU0126 strain. Porins are the channel-forming protein that helps in the transport of molecules across the selectively permeable bacterial membrane bilayer. Mutations or changes in the porin proteins, such as loss or modification of the size of porin or lower expression result in the limited diffusion of β-lactams, fluoroquinolones, tetracycline and chloramphenicol [64]). Many of the genes coding for porins had both positive and negative-fold change and reduced differential expression among both the A. baumannii ATCC 19606 and JU0126 strains treated with eravacycline like the carbapenem susceptibility porin CarO (-4- and -0.2-fold change), OmpW family protein (-3- and -8-fold change), outer membrane porin OprD family (5- and -4-fold change) and OmpA family protein (-0.06 and 3-fold change). This reduction in the expression of these porins signifies their role in conferring resistance by decreasing the antibiotic entry into cell.
Although β-lactamase enzyme production is not related to the eravacycline resistance, few classes of β-lactamase were noted to have both positive and negative-fold change. The genes for enzymes MBL-fold metallohydrolase had 5-fold change and 4-fold change, OXA-51 family carbapenem-hydrolysing class-D β-lactamase OXA-259 with 1 and -0.4-fold change and class C extended-spectrum β-lactamase ADC-26 with -1-fold and 7-fold change for A. baumannii ATCC 19606 and JU0126 treated strains, respectively.
OMVs proteins with function pertaining to stress and resistance
The proteins involved with virulence, stress response and antibiotic resistance expressed in the OMVs of eravacycline induced A. baumannii strains of ATCC 19606 and JU0126 were compared with the uninduced controls with respect to their log2-fold change (only proteins with more than 2 log2-fold change are mentioned below). Many proteins especially ribosomal proteins had more than 2 log2-fold change in the expression in both the ATCC 19606 and clinical strain JU0126 and apart from that chaperons, OMP and resistance-conferring proteins were observed. Prior studies have also reported many OMP [65], [66] and resistance-conferring proteins expressed in OMVs of antibiotic-treated strains, our study identified many OMP and antibiotic resistance-related proteins from both A. baumannii ATCC 19606 and JU0126. In the ATCC 19606 strain, highest log2-fold change was for OmpA family protein (5.66), followed by Omp38 (4.43), β-lactamase (3.40), OprD family (2.91) and putative acriflavine resistance protein A (2.30). Other proteins pertaining to virulence, stress and bacterial survival with more than 2 log2-fold change were copper-exporting ATPase (9.65) which is a copper tolerance protein, toluene tolerance protein Ttg2D (8.87), TonB-dependent siderophore receptor (6.69), 50S ribosomal proteins L14, L6, L4, L19, L16, L29 and L2 (log2-fold change range from 2 to 6), 30S ribosomal proteins S11, S3 and S7 (log2-fold change 3–4), peptidases S41 family (6.53), peptidoglycan-associated protein (6.0), type IV pilus biogenesis/stability protein PilW (4.94), type VI secretion protein, EvpB/VC_A0108 family (3.15), translation initiation factor IF-3 (4.12), TolB belonging to the Tol–Pal peptidoglycan-associated lipoprotein system protein (3.34), chaperone protein HscA homolog that belongs to the heat shock protein 70 family (2.75) and vacJ-like lipoprotein. There were just two proteins associated with resistance showing more than 2 log2-fold change in the OMV proteome of A baumannii JU0126 strain, β-lactamase protein, and major facilitator family transporter. However, many stress response proteins, virulence, and survival proteins were expressed with more than 2 log2-fold change in JU0126. The same as A. baumannii ATCC 19606, ribosomal protein abundance was very significantly high noting that the strains were induced resistance to eravacycline. 30S ribosomal proteins S5, S4, S2, S3, S9 ranged from 2 log2-fold change to 9 log2-fold change and the 50S ribosomal proteins L4, L6, L2, L1, L18, L16, L28, L10 with log2-fold ranging between 4 and 8. Other proteins like toluene tolerance protein Ttg2D, Tol–Pal system protein TolB, gamma-glutamyl transferase, acetyl-CoA C-acetyltransferase, transcription termination factor Rho, YqaJ viral recombinase family protein, signal recognition particle protein, TonB-dependent siderophore receptor, and peptidases M48, S41 were expressed with more than 2 log2-fold change in the eravacycline induced strains.
Inconsistency in the expression patterns of OMVs Proteins in comparison to the bacterial whole gene expression profiles
The overall results from the comparison of the two expression profiles, the protein, and the RNA were with a very low correlation coefficient. Some of the ribosomal proteins were upregulated in both RNA and OMV proteome expression profiles. The expression of ribosomal proteins in the OMV proteome can be supported by reports on the presence of RNAs and the proteins involved in their synthesis. Sjöström et al. (2015) reported for the first time that RNAs were involved with bacterial OMVs [67]. Other proteins with a correlation between mRNA and protein expression include dihydrolipoamide acetyltransferase, DUF4142 domain-containing protein, class C extended-spectrum β-lactamase ADC-26 and a hypothetical protein. In the strain A. baumannii ATCC 19606, although many ribosomal proteins showed upregulation in their expression, linear correlation of both mRNA and protein expression was seen only in, copper-translocating P-type ATPase, methylmalonate-semialdehyde dehydrogenase (CoA acylating), adenosine deaminase and gamma-glutamyltransferase family g-protein. The low correlation of the mRNA and protein components based on the log2-fold change comparison suggests that proteins in OMVs are selectively enriched, transported from the bacterial cell and/or due to wide range of regulatory mechanisms involved in the post-transcriptional level [68]. A poor correlation of similar comparison was reported by Yun et al. (2018) in their study of proteins in OMVs and protein fractions from bacterial cell membranes. They have mentioned the reason to be that proteins in the OMVs are differentially selected and sorted from the host bacteria.
Enriched biological pathways
PPI networks from commonly expressed gene/proteins from both transcriptome and OMVs proteome of ATCC 19606 and JU0126 strains were constructed. Pathways that were found enriched were significantly ironically related to transcription and RNA synthesis, owing to the fact that the bacterium was grown in an eravacycline stressed environment and the subsequent induced resistance.