Magnified regions of the upregulated proteins identified by

Magnified regions of the upregulated proteins identified by MALDI-TOF are shown in Fig. 2. The identified proteins were phosphoglycerate kinase, fructose-bisphosphate aldolase class II, alcohol dehydrogenase, phosphoglyceromutase, 6-phosphogluconolactonase, malate dehydrogenase, alkyl hydroperoxide reductase subunit C, DNA protection during starvation protein, 2,5-diketogluconate reductase A, cysteine synthase A, LysM domain/BON superfamily protein, 60kDa chaperonin GroEL, small heat shock protein, putative non-heme chloroperoxidase and putative intracellular proteinase. Two spots in the gel were identified as the same protein, malate dehydrogenase. The level of overexpression is represented as the densitometric ratio in Table 1. The density of spots in the two gels was calculated by Quantity One® (Supplementary Fig. S1; Supplementary Table S1, Table S2). The protein LysM domain/BON superfamily protein was expressed by >12-fold in the drug-treated condition.
Discussion The emergence of resistance to carbapenems in bacteria has increased significantly in the past few decades. In this study, we sought to identify proteins playing a pivotal role in resistance mechanisms of K. pneumoniae NP6, besides KPC-type β-lactamase. To gain an insight into the post-treatment physiological changes occurring in bacteria, the whole-cell lysates of NP6 growing in exponential phase in the presence and absence of meropenem were analysed and compared. The bacterial culture was treated with a sub-MIC (0.5×MIC) concentration of the antibiotic. The current data revealed increased expression of stress-induced proteins (60kDa chaperonin GroEL and small heat shock protein). Under stress conditions in bacteria, these proteins are known to play an important role in preventing protein aggregation by aiding proper protein folding as well as refolding of misfolded proteins [20]. In this study, many proteins were overexpressed following meropenem treatment, so apparently chaperonin GroEL and small heat shock protein are overexpressed to ensure proper folding of a multitude of different proteins during their hasty production to counter the effects of the antibiotic. Previous studies have revealed that bactericidal DNQX disodium salt stimulate genetic and biochemical changes in bacteria leading to the production of highly deleterious reactive oxygen species (ROS) [21]. Thus, bacteria showing tolerance to bactericidal antibiotics must possess well-orchestrated mechanisms to detoxify the stress and to repair the damage caused by ROS [22]. In this study, four different enzymes were identified that appear to play an important role in detoxifying the effects of ROS. Two of these proteins, previously well documented to be associated with resistance against bactericidal antibiotics, were identified as alkyl hydroperoxide reductase subunit C (AhpC) and DNA protection during starvation protein (Dps). AhpC, along with another flavoprotein AhpF, comprise alkyl hydroperoxide reductase (AhpR), which is the most studied bacterial scavenging enzyme against organic hydroperoxides [23]. AhpR is involved in NAD(P)H-dependent reduction of organic peroxides to alcohols [24]. Dps is a non-specific DNA-binding protein that protects DNA from oxidative stress [25]. Studies in E. coli have shown the role of Dps in oxidative damage protection especially against peroxides during exponential phase [26]. In our case, both AhpC and Dps are assumed to play the same role of protection against the deleterious effects of meropenem in bacterial cells and hence their expression levels are increased. Spot 12 corresponded to 6-phosphogluconolactonase, which is an enzyme of the pentose phosphate pathway (PPP). The PPP yields reducing equivalents in the form of NADPH that help in maintaining the redox potential inside cells. Again, overexpression of this enzyme indicates its potential role in protecting bacterial cells from oxidative stress in the presence of the antibiotic meropenem. Another protein, identified as 2,5-diketo-d-gluconate reductase, was found to have increased expression. This enzyme catalyses the conversion of 2,5-diketo-d-gluconic acid into 2-keto-l-gluconic acid (direct precursor of l-ascorbic acid/vitamin C). Upregulation of this enzyme might be indicative of some involvement of ascorbic acid in the bacterial resistance mechanism, although this aspect requires further exploration. Many studies have shown that vitamin C plays a vital role as a low-molecular-weight antioxidant in plasma and it demonstrates synergistic effects with other antioxidants [27]. Ascorbate has been shown to effectively scavenge superoxide radical anions, hydrogen peroxides, hydroxyl radicals and singlet oxygen [28].