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The Golgi apparatus is an organelle that mediates modifications, sorting, and transport of proteins and lipids. Golgins are a group of proteins with coiled-coil structures that localize to the Golgi and are thought to function as tethers to facilitate the docking of vesicles, Rab GTPases, and cytoskeleton components to the Golgi stack. Giantin is the longest golgin and has been thought to function as a tether for COPI vesicles along with other golgins, such as p115 and GM130. Contrary to our expectation that the loss of the tether will result in an increase in untethered COPI vesicles in the cytoplasm, our electron microscopy observations showed that the fenestrae normally present in Golgi cisternae were reduced upon Giantin knockdown. We also found that this structural change is accompanied by altered secretion of cargo proteins and cell surface glycosylation. These results indicate that there exists a correlation between Golgi structural changes caused by the loss of Giantin and Golgi function. Here, we describe electron tomography methods for the detection of structural changes in the Golgi.

The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.

Cefiderocol is a novel siderophore cephalosporin antibiotic exhibiting activities against carbapenem-resistant Gram-negative bacteria including Pseudomonas aeruginosa and Enterobacteriaceae. Drug efflux pumps are reportedly involved in both intrinsic and acquired drug resistance, although their role in bacterial cefiderocol susceptibility remains poorly understood. In this study, we investigated how drug efflux pumps contribute to bacterial cefiderocol susceptibility using the efflux pump(s)-deficient and overexpressing strains of P. aeruginosa, Escherichia coli, and Salmonella enterica. We observed that the mexAB-oprM-deficient P. aeruginosa mutant displayed increased cefiderocol susceptibility compared to the wild-type strain. The overexpression of mexAB-oprM or mexXY-oprM in the mexAB-oprM-deficient mutant increased the MIC value of cefiderocol. Furthermore, the pump inhibitor phenylalanine-arginine β-naphthylamide increased cefiderocol susceptibility in wild-type P. aeruginosa whereas it did not affect the susceptibility of the mexAB-oprM-deficient mutant. These data indicate that the MexAB-OprM drug efflux system contributes to the intrinsic cefiderocol resistance of P. aeruginosa. In addition, MexXY-OprM partially complemented the function of MexAB-OprM in the cefiderocol susceptibility, when expressed.

Multidrug resistance (MDR) in bacteria can be caused by the over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily of proteins. These intrinsic or acquired pumps can export a wide range of antibiotics. Recently, amino acid substitutions within these pumps have been observed in resistant clinical strains. Among others, two of these worrying gain-of-function mutations are R717L and R717Q in the proximal binding pocket of efflux pump AcrB (AcrB-Sa) found in azithromycin-resistant Salmonella enterica spp. We investigated the ramifications of these (and other) mutations in phylogenetically closely related AcrB from Escherichia coli (AcrB-Ec). We found that AcrB-Ec harboring Arg717 substitutions were significantly more effective in exporting all tested macrolides, with an up to 8-fold increase in the minimum inhibitory concentration (MIC) values (from 16 to 128 μg/mL for azithromycin). Interestingly, gain-of-function was also seen for fluoroquinolones (2-fold higher MICs), while there was a consistent loss-of-function for the export of novobiocin and β-lactam cloxacillin (2-fold lower MICs), pointing to a protein adaptation, which simultaneously partly compromised the efflux ability of other compounds with different molecular properties. Disk diffusion susceptibility testing corroborated the findings, as the R717Q and R717L mutant strains had significantly smaller inhibition zones for macrolides and fluoroquinolones and a larger inhibition zone for novobiocin, compared to the wild type. The spread and independent emergences of these potent efflux pump mutations highlight the necessity of control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors.ImportanceThe over-expression of multidrug efflux pumps is a major cause of multidrug-resistant bacteria. The emergence of gain-of-function mutations in these pumps is additionally worrying, as these render last-resort antibiotic treatment options ineffective. The Arg717-mutations in related Salmonella Typhi and Paratyphi A AcrB caused azithromycin-resistant strains, with resistance levels past breakpoints. This is worrying, as azithromycin is a last-resort antibiotic after fluoroquinolones to treat typhoid and paratyphoid fever. Our findings that the same substitutions in closely related Escherichia coli AcrB cause an increase in MICs for both fluoroquinolones and macrolides is worrying as it shows the effect of one amino acid substitution on a multitude of treatment options. Additionally, the finding that the substitutions negatively impact the export of other drugs, such as β-lactam cloxacillin, suggests that treatment with multiple antibiotics may mitigate resistance and improve treatment. Our findings also imply the urge to develop novel antibiotics and inhibitors.

The emergence of bacteria that are resistant to antibiotics is common in areas where antibiotics are used widely. The current standard procedure for detecting bacterial drug resistance is based on bacterial growth under antibiotic treatments. Here we describe the morphological changes in enoxacin-resistant Escherichia coli cells and the computational method used to identify these resistant cells in transmission electron microscopy (TEM) images without using antibiotics. Our approach was to create patches from TEM images of enoxacin-sensitive and enoxacin-resistant E. coli strains, use a convolutional neural network for patch classification, and identify the strains on the basis of the classification results. The proposed method was highly accurate in classifying cells, achieving an accuracy rate of 0.94. Using a gradient-weighted class activation mapping to visualize the region of interest, enoxacin-resistant and enoxacin-sensitive cells were characterized by comparing differences in the envelope. Moreover, Pearson’s correlation coefficients suggested that four genes, including lpp, the gene encoding the major outer membrane lipoprotein, were strongly associated with the image features of enoxacin-resistant cells.

Multidrug efflux pumps play an important role in bacterial multidrug resistance by actively excreting antibiotics. The ATP-binding cassette-type drug efflux pump MacAB was originally reported as a macrolide-specific pump. MacAB is also known to be required for the virulence of Salmonella enterica serovar Typhimurium following oral infection in mice. Here, we performed a screening of inhibitors of Salmonella MacAB and found a compound that increased the susceptibility of a MacAB-expressing strain to macrolides. It was previously reported that MacAB is required to resist peroxide-mediated killing in vitro and that a supernatant of wild-type Salmonella rescues the growth defect of a macAB mutant in H2 O2 . In this study, we also found that the MacAB inhibitor reduced the ability of the supernatant to rescue Salmonella cells in H2 O2 . This compound could lead to a better understanding of the function of MacAB.

The structure determination of organic compounds is desirable for the development of medicines, aroma chemicals, and agricultural chemicals. However, the crystallization of organic compounds is often troublesome, because crystallization requires a relatively large quantity of high purity compounds and crystallization trials often need to be performed repetitively using different conditions. Some proteins are known to be able to bind to various organic compounds. The multidrug-resistance regulator protein RamR is one such protein. We have developed a structure determination method for organic compounds using RamR. RamR bound to organic compounds, including one compound that was not a known ligand for RamR, and the structures of the complexes were successfully determined. Because the RamR crystal is hydrophilic, this method may be useful for compounds that cannot be handled by the crystalline sponge method.

Increased expression of efflux pumps is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa, and treatment with inhibitors of active efflux pumps seems an attractive strategy to combat with multidrug resistance. Assays using ethidium bromide (EtBr), which accumulates by binding to nucleic acids, are often employed to assess the efficacy of efflux pump inhibitors (EPIs). However, few studies have reported on assays using other nucleic acid dyes.We used different classes of EPIs for MexAB- or MexXY-OprM to measure the accumulation of various fluorescent dyes, including SYBR Safe, AtlasSight, and GelGreen.Escherichia coli MG1655ΔacrBΔtolC strain harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of P. aeruginosa were constructed. Then, the accumulation of the above-mentioned nucleic acid dyes and EtBr was measured to assess the efflux ability in the presence and absence of EPIs (MexAB-OprM-specific inhibitor of pyridopyrimidine derivative [ABI-PP], berberine, non-specific inhibitor of phenylalanine-arginine β-naphthylamide [PAβN], and protonophore of carbonyl cyanide m-chlorophenyl hydrazone [CCCP]).Decreased accumulations of nucleic acid dyes were observed in strains with pABM or pXYM compared with the parental strain. ABI-PP or berberine addition significantly increased the accumulation of any nucleic acids in the strains with the specific pumps. PAβN or CCCP addition showed increased accumulation of almost all dye in strains with pABM or pXYM. However, the inhibition patterns of EPIs differed according to the nucleic acid dyes used.Accumulation assays for EPIs were suitable to evaluate EPI candidates using various nucleic acid dyes.

RND-type multidrug efflux pumps have two voluminous multisite drug-binding pockets named the proximal and distal binding pocket. High- and low-molecular-mass drugs bind to these proximal and distal pocket, respectively. Here, we report the crystal structures of MexB of Pseudomonas aeruginosa bound with high-molecular-mass compounds. Contrary to the expectations, lauryl maltose neopentyl glycol (LMNG, MW 1,005), which is a surfactant larger than the proximal pocket-binding drugs, was found to bind to the distal pocket: one of the two hydrophobic alkyl chains was inserted into the hydrophobic pit, which is the binding site of the efflux pump inhibitor ABI-PP. LMNG is a substrate of the MexAB-OprM system and competitively inhibits the export of other substrates by this system. However, LMNG does not inhibit the export of other substrates by the inhibitor-binding-pit mutant F178W, which retains the export activity of LMNG. The crystal structure of this mutant suggested that the alkyl chain of LMNG could no longer be inserted into the pit because of steric hindrance. We also determined the crystal structure of MexB containing the high-molecular-mass compound neopentyl glycol derivative C7NG (MW 1,028), the binding site of which overlapped with LMNG in the distal pocket, indicating that whether a substrate binds to the distal or proximal pockets is controlled not only by its molecular weight but also by its individual molecular characteristic.

Multidrug resistance in Gram-negative bacteria can arise by the over-expression of multidrug efflux pumps, which can extrude a wide range of antibiotics. Here we describe the ancestral Haemophilus influenzae efflux pump AcrB (AcrB-Hi). We performed a phylogenetic analysis of hundreds of RND-type transporters. We found that AcrB-Hi is a relatively ancient efflux pump, which nonetheless can export the same range of antibiotics as its evolved colleague from Escherichia coli. AcrB-Hi was not inhibited by the efflux pump inhibitor ABI-PP, and could export bile salts weakly. This points to an environmental adaptation of RND transporters. We also explain the sensitivity of H. influenzae cells to β-lactams and novobiocin by the outer membrane porin OmpP2. This porin counterbalances the AcrB-Hi efflux by leaking the drugs back into the cells. We hypothesise that multidrug recognition by RND-type pumps is not an evolutionarily acquired ability, and has been present since ancient promiscuous transporters.

Multidrug resistance in Gram-negative bacteria can arise by the over-expression of multidrug efflux pumps, which can extrude a wide range of antibiotics. Here we describe the ancestral Haemophilus influenzae efflux pump AcrB (AcrB-Hi). We performed a phylogenetic analysis of hundreds of RND-type transporters. We found that AcrB-Hi is a relatively ancient efflux pump, which nonetheless can export the same range of antibiotics as its evolved colleague from Escherichia coli. AcrB-Hi was not inhibited by the efflux pump inhibitor ABI-PP, and could export bile salts weakly. This points to an environmental adaptation of RND transporters. We also explain the sensitivity of H. influenzae cells to β-lactams and novobiocin by the outer membrane porin OmpP2. This porin counterbalances the AcrB-Hi efflux by leaking the drugs back into the cells. We hypothesise that multidrug recognition by RND-type pumps is not an evolutionarily acquired ability, and has been present since ancient promiscuous transporters., Zwama et al. demonstrate that the AcrB efflux pump from H. influenzae (AcrB-Hi) is phylogenetically far removed from AcrB in E. coli but show similar specificity. They also show that efflux is counteracted by leakage of some antibiotics back into the bacterial cell through the OmpP2 channel which can explain H. influenzae sensitivity to these compounds.

Salmonella Enteritidis has developed the potential to contaminate eggs by surviving in the antimicrobial environment of the hen's egg white. This has led to a worldwide pandemic of foodborne salmonellosis infections in humans due to the consumption of contaminated eggs and egg-derived products. The molecular mechanisms of Salmonella Enteritidis egg white survival are not fully clear. Using in vivo expression technology and promoter-reporter fusions we showed that the promoter of the tolC gene, encoding the TolC outer membrane channel that is used by multidrug efflux pumps to export harmful molecules and to secrete bacterial products, is activated by egg white at the chicken body temperature. Using a Salmonella Enteritidis tolC deletion mutant we showed that TolC has an important role in egg white survival. Chromatographic separation techniques and subsequent testing of antimicrobial activities of separated egg white fractions led to the identification of ovotransferrin as the egg white antimicrobial factor which is capable of inhibiting growth of a tolC deletion strain but not the wild type strain. We provide evidence that TolC protects Salmonella Enteritidis against ovotransferrin-mediated growth inhibition in egg white.

The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB -deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCE The structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB -deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrAB-TolC-mediated multidrug export.

Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.

Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

Multidrug efflux pumps are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. ABI-PP (a pryridopyrimidine derivative) is a MexB-specific inhibitor that does not inhibit MexY; MexB and MexY are principal pumps in Pseudomonas aeruginosa. We previously found that drugs were exported through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of pumps. ABI-PP binds tightly to a narrow pit located in the distal pocket and sterically hinders the functional rotation. Phenylalanine is located at the edge of this pit in MexB and contributes to the tight binding of the inhibitor molecule. On the other hand, the voluminous side chain of tryptophan located at the corresponding position in MexY prevents inhibitor binding. For the development of universal inhibitors of MexB and MexY, it is important to avoid the steric hindrance of tryptophan in MexY. Now we are developing clinically useful inhibitors on the basis of the structural information obtained. Started from the ABI-PP structure, we designed many compounds that can bind to the inhibitor-binding pits of MexB and MexY. Some of designed compounds were actually synthesized and their inhibitory activity determined. Finally, we obtained some lead compounds that showed complete prevention of the growth of strains expressing MexB and MexY with low concentrations of antibiotics.

Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi.

The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g., amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the four other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains. Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important.

During infection, Salmonella senses and responds to harsh environments within the host. Persistence in a bile-rich environment is important for Salmonella to infect the small intestine or gallbladder and the multidrug efflux system AcrAB-TolC is required for bile resistance. The genes encoding this system are mainly regulated by the ramRA locus, which is composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, whose encoding gene is itself transcriptionally repressed by RamR. RamR recognizes multiple drugs; however, the identity of the environmental signals to which it responds is unclear. Here, we describe the crystal structures of RamR in complexes with bile components, including cholic acid and chenodeoxycholic acid, determined at resolutions of 2.0 and 1.8 Å, respectively. Both cholic and chenodeoxycholic acids form four hydrogen bonds with Tyr59, Thr85, Ser137 and Asp152 of RamR, instead of π–π interactions with Phe155, a residue that is important for the recognition of multiple compounds including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6 G. Binding of these compounds to RamR reduces its DNA-binding affinity, resulting in the increased transcription of ramA and acrAB-tolC. Our results reveal that Salmonella senses bile acid components through RamR and then upregulates the expression of RamA, which can lead to induction of acrAB-tolC expression with resulting tolerance to bile-rich environments.

A directly accessible femtoliter droplet array as a platform for single-cell detection and collection of persister bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single cells, long-term culture and observation of single cells in droplets, and collection of identified persisters from single droplets are described in detail.

Salmonella species are causative organisms of salmonellosis, and the prevalence of multidrug-resistant Salmonella has increased dramatically. These multidrug-resistant isolates have been found in both humans and animals and thus pose a major public health concern. Drug resistance in Salmonella has been shown to be largely attributable to multiple target gene mutations and to active efflux by pumps. At least ten drug efflux system genes in the genome of this organism have been experimentally identified to date, and some efflux pump genes encoded in plasmids have been also identified. This chapter describes the drug resistance and virulence roles of efflux pumps and their regulation in Salmonella.

A directly accessible femtoliter droplet array as a platform for single-cell detection and collection of persister bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single cells, long-term culture and observation of single cells in droplets, and collection of identified persisters from single droplets are described in detail.