Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.     

Modulatory Effects of Vasoactive Intestinal Peptide on Intestinal Mucosal Immunity and Microbial Community of Weaned Piglets Challenged by an Enterotoxigenic Escherichia coli (K88)

Toll-like receptors (TLRs) recognize microbial pathogens and trigger immune response, but their regulation by neuropeptide-vasoactive intestinal peptide (VIP) in weaned piglets infected by enterotoxigenic Escherichia coli (ETEC) K88 remains unexplored. Therefore, the study was conducted to investigate its role using a model of early weaned piglets infected by ETEC K88. Male Duroc×Landrace×Yorkshire piglets (n = 24) were randomly divided into control, ETEC K88, VIP, and ETEC K88+VIP groups. On the first three days, ETEC K88 and ETEC K88+VIP groups were orally administrated with ETEC K88, other two groups were given sterile medium. Then each piglet from VIP and ETEC K88+VIP group received 10 nmol VIP intraperitoneally (i.p.) once daily, on day four and six. On the seventh day, the piglets were sacrificed. The results indicated that administration of VIP improved the growth performance, reduced diarrhea incidence of ETEC K88 challenged pigs, and mitigated the histopathological changes of intestine. Serum levels of IL-2, IL-6, IL-12p40, IFN-γ and TNF-α in the ETEC K88+ VIP group were significantly reduced compared with those in the ETEC group. VIP significantly increased IL-4, IL-10, TGF-β and S-IgA production compared with the ETEC K88 group. Besides, VIP could inhibit the expression of TLR2, TLR4, MyD88, NF-κB p65 and the phosphorylation of IκB-α, p-ERK, p-JNK, and p-38 induced by ETEC K88. Moreover, VIP could upregulate the expression of occludin in the ileum mucosa compared with the ETEC K88 group. Colon and caecum content bacterial richness and diversity were lower for pigs in the ETEC group than the unchallenged groups. These results demonstrate that VIP is beneficial for the maturation of the intestinal mucosal immune system and elicited local immunomodulatory activities. The TLR2/4-MyD88 mediated NF-κB and MAPK signaling pathway may be critical to the mechanism underlying the modulatory effect of VIP on intestinal mucosal immune function and bacterial community.     

Episome-carried Surface Antigen K88 of Escherichia coli II. Isolation and Chemical Analysis

The K88 antigen was carried by episomal transfer to D282, a nonmotile Escherichia coli strain without K antigen. D520, obtained by this episomal transfer, was used for the extraction of K88 antigen. It was shown by the agar gel precipitation technique that some K88 antigen was released from D520 into suspending aqueous medium. The amount of liberated material was increased by gentle heating (60 C) or treatment in a Waring Blendor. The antigen was obtained from the extracts in a purified form by making use of its insolubility between pH 3.5 and 5.5 and of its high sedimentation rate (S(0) (20,w) = 36.7S). The homogeneity of the material was demonstrated by agar gel precipitation with D520 antiserum, by analytical ultracentrifugation, and by moving-boundary electrophoresis. Chemical analysis revealed that K88 is a pure protein containing all the common amino acids with the exception of cysteine-cystine. Purified K88 selectively precipitated the K88 antibodies from D520 antiserum and was shown to be immunogenic in rabbits.     

Inhibition of adhesion of enterotoxigenic Escherichia coli K88 by soya bean tempe

AIMS: Tempe is a traditional fungal fermented food made from soaked and cooked soya beans. It has been associated with antidiarrhoeal characteristics. This study investigated potential inhibitory effects of tempe on enterotoxigenic Escherichia coli (ETEC) K88. METHODS AND RESULTS: Soya beans were soaked, cooked and subsequently fermented using several Rhizopus spp. Water-soluble filter-sterile extracts were tested for their ability to inhibit growth of E. coli and several indicator microorganisms and to inhibit adhesion of ETEC K88. Antimicrobial activity was found against Bacillus stearothermophilus only. ETEC K88-induced haemagglutination of hamster red blood cells was strongly inhibited by a number of tempe extracts and hardly by the cooked soya bean extract. Furthermore, several tempe extracts were able to inhibit adhesion of ETEC K88 to piglet small intestinal brush-border membranes. CONCLUSIONS: Tempe appeared to interfere with ETEC K88 adhesion rather than showing growth inhibitory properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that tempe could exert an antagonistic effect against ETEC through inhibition of adhesion and might therefore have a protective effect against ETEC K88 infection in pigs. Hence, tempe could have potential to use as a feed supplement in the diet of weaned piglets.     

Exopolysaccharides Synthesized by Lactobacillus reuteri Protect against Enterotoxigenic Escherichia coli in Piglets

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter⁻¹ of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated that E. coli is the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (P = 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescent in situ hybridization analysis demonstrated that reuteran significantly (P = 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.     

Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells

Aims:  This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action.
Methods and Results:  Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells.
Conclusions:  Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds.
Significance and Impact of the Study:  The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases.     

Oral immunization of mice with plant-derived fimbrial adhesin FaeG induces systemic and mucosal K88ad enterotoxigenic Escherichia coli-specific immune responses

The importance of adhesins in pathogenicity has resulted in them being useful targets in the defense against bacterial infections. To produce edible vaccines against piglet diarrhea caused by enterotoxigenic Escherichia coli (ETEC), plants were genetically engineered to produce recombinant fimbrial adhesin FaeG. To evaluate the efficacy of the edible vaccine FaeG in mice, the soluble protein extracts were examined by about 15 microg recombinant FaeG for each oral immunization dose per mouse. After four doses of vaccination, both IgG and IgA antibodies specific to K88ad fimbriae were elicited in serum, and specific IgA antibodies were also evoked in feces of the immunized mice. Moreover, visible K88ad ETEC agglutination by the specific serum from the immunized mice was observed, implying the antibody was highly specific and effective. Results from an in vitro villous-adhesion assay further confirmed that serum antibodies of the immunized mice could inhibit K88ad ETEC from adhering to pig intestinal receptors, further demonstrating the oral immune efficacy of the plant-derived FaeG. This study provides a promising, noninvasive method for vaccinating swine by feeding supplements of transgenic plant. Moreover, the low cost and ease of delivery of this edible ETEC vaccine will facilitate its application in economically disadvantaged regions.     

Complete release of adenosine deaminase from mouse lymphocytes stabilized by low-pH acetate

Complete release of adenosine deaminase from mouse lymphocytes takes place when intact cells are stabilized by low-pH acetate buffer. Both the low pH and the acetate affect the enzyme extraction markedly. At pH 5.0 all the adenosine deaminase activity detectable in the whole cell homogenates is released into the acetate buffer in very few minutes, with a total amount of 2% protein being extracted. The complete extraction of the enzyme activity is never observed when, at pH 5.0, the acetate is replaced by glutamate, citrate, succinate or maleate and only 45% and 15% of the adenosine deaminase activity is extracted by the acetate at pH 6.0 and 7.0, respectively. The breakdown of adenosine by the enzyme activity extracted from the stabilized cells is due to deamination alone, since inosine is the only product of the catalyzed reaction and its formation is completely inhibited by coformycin, a selective inhibitor of adenosine deaminase. The enzyme extracted shows a specific activity 50-times higher than that found in the crude homogenates, and a substantial purification of the enzyme extracted is achieved by a single Sephadex G-100 gel filtration.     

动物源产肠毒素大肠杆菌(ETEC)黏附素研究进展

动物源产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是引起动物(尤其是幼龄动物)腹泻的主要病原菌。已知黏附素和肠毒素是ETEC中两种重要的毒力因子,在致病性中两者缺一不可。其中黏附素结合到宿主易感肠上皮细胞是ETEC感染的第一步,也是最重要的关键步骤。动物源ETEC的菌毛黏附素主要包括K88、K99、987P、F18、F17和F41等。人们从20世纪60年代就开始了ETEC菌毛黏附素的相关研究,包括菌毛的基因、结构组成、生物合成、菌毛表达的调控机制以及黏附素和宿主受体相互作用等,这些研究基础有助于我们深入了解ETEC病原菌的感染机理;并且在疾病诊断和新疫苗的开发中具有重大意义。     

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

[目的]揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系.[方法]O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测.[结果]通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以0149、0107、0139、093和091为主,共133株,占定型菌株的57.1%;拥有est Ⅰ、estⅡ、elt、stx2e和eae A基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,0149血清型与est Ⅰ或estⅡ elt密切相关,在F18菌株中,0107血清型与est Ⅰ或estⅡ、0139血清型与stx2e紧密相关.依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%.通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以0149、0107、093和098等血清型为主,0149:K88菌株主要与estⅡ或estⅡ elt肠毒素相关,0107:F18菌株主要与estⅡ相关,093和098血清型菌株主要与estⅡ肠毒素相关;STEC菌株以0139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以0107:F18和0116:F18血清型为主,主要与est Ⅰ stx2e或estⅡ stx2e密切相关,ETEC/AEEC菌株以091和0107血清型为主,全部拥有肠毒素est Ⅰ和紧密素基因.[结论]我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂.     

Isolation of K88 Antigen Variants (ab,ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes

Fimbriae isolation by means of thermal shock was applied to fifteen K88-positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes O8:K87, O32, O45, O138:K81, O141:K85, O147:K89, O149:K91, and O157, as well as to ten K88-positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the O149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris-HCl for isolation of K88 antigen. The molecular weights by SDS-PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant.     

Polyamine Binding to Proteins in Oat and Petunia Protoplasts

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozenthawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.     

Protective immune response of bacterially-derived recombinant FaeG in piglets

FaeG is the key factor in the infection process of K88ad enterotoxigenic Escherichia coli(ETEC) fimbrial adhesin. In an attempt to determine the possibility of expressing recombinant FaeG with immunogenicity for a new safe and high-production vaccine in E. coli, we constructed the recombinant strain, BL21 (DE3+K88), which harbors an expression vector with a DNA fragment of faeG, without a signal peptide. Results of 15% SDS-polyacrylamide slab gel analysis showed that FaeG can be stably over-expressed in BL21 (DE3+K88) as inclusion bodies without FaeE. Immunoglobulin G (IgG) and M (IgM) responses in pregnant pigs, with boost injections of the purified recombinant FaeG, were detected 4 weeks later in the sera and colostrum. An in vitro villius-adhesion assay verified that the elicited antibodies in the sera of vaccinated pigs were capable of preventing the adhesion of K88ad ETEC to porcine intestinal receptors. The protective effect on the mortality rates of suckling piglets born to vaccinated mothers was also observed one week after oral challenge with the virulent ETEC strain, C83907 (K88ad, CT+, ST+). The results of this study proved that the adhesin of proteinaceous bacterial fimbriae or pili could be overexpressed in engineered E. coli strains, with protective immune responses to the pathogen.     

Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets

The protective effects of egg-yolk antibodies obtained from hens immunized with fimbrial antigens from a local strain (Escherichia coli K88+ MB, Manitoba, Canada) of K88+ piliated enterotoxigenic E. coli (ETEC) were evaluated in 3- and 21-day-old piglets in which ETEC diarrhea was induced and also in early-weaned piglets in a commercial farm. The results demonstrated that the E. coli K88+ MB-induced diarrhea in 3-day-old piglets was cured 24 h after treating with egg-yolk antibodies while those treated with egg-yolk powder from conventional hens continued to have diarrhea and 62.5% of them died of severe diarrhea. For 21-day-old weaned piglets, those fed egg-yolk antibodies had transient diarrhea, positive body weight gains and 100% survival during the period of the experiment, whereas control piglets that were treated with placebo had severe diarrhea and dehydration and some died within 48 h after infection. In the field trial, the incidence and severity of diarrhea of 14-18-day-old weaned piglets fed egg-yolk antibodies were much lower than in those fed a commercial diet containing an antibiotic. These results indicate that the neonatal and early-weaned piglets that received the egg-yolk antibodies were protected against ETEC infection.     

抗大肠埃希氏菌K88ab,K88ac和K88ad特异单克隆抗体

A panel of twelve hybridoma cell lines, secreting specific antibodies to K88 adhesin antigens of enterotoxigenic Escherichia coli (ETEC) were established from eight separate fusions between mouse myeloma cell line Sp 2/0-Ag-14 and spleen cells from mice immunized with purified K88 antigens. Among the 12 monoclonal antibodies (MCA), K-A, K-35, K-11, and K-15 were K88a specific and reacted with all K88 adhesin bearing Escherichia coli strains tested, whatever K88ab, K88ac or K88ad they might be, as shown either in enzyme-linked immunosorbent assay (ELISA) or in direct agglutination test, whereas K32, K-4, and K-3 were specific for G88ab, K88ac, and K88ad respectively. The antigen patterns of 33 K88 bearing Escherichia coli strains covering 3 serotypes of K88ab, K88ac, and K88ad were analyzed by the use of these MCAs. The preliminary results showed that all Escherichia strains with the same serotype of K88 antigen shared at least one common type-specific antigenic determinant, that K88ad and K88ac strains enjoyed one common antigenic determinant that did not exist on K88ab strains, and that there were a few K88 antigenic determinants that appeared only on limited Escherichia coli strains of the same K88 serotype.     

乳杆菌对致病性大肠杆菌K88和O138体外存活抑制的动力学研究

研究了一株分离自断奶仔猪小肠黏膜的肠乳杆菌L1（Lactobacillus intestinalis）体外发酵特性,及其代谢产物对病原性大肠杆菌Escherichia coli K88和O138存活的影响。体外发酵结果表明：发酵12h后,L,菌液pH值迅速降至3．90,并产生大量乳酸,为104．08mmol／L。L1菌株代谢产物对K88和O138体外生长抑制的动力学研究表明：L1菌株代谢产物对K88和O138存活具有很强的抑制作用;L1菌株发酵液与含相同浓度乳酸的自制培养液比较结果表明：乳酸在L1菌株代谢物对K88和O138存活抑制中发挥了主要作用：K88和O138对pH4．5的MRS培养液具有一定的耐受能力。     

乳杆菌对致病性大肠杆菌K88和O138体外存活抑制的动力学研究

研究了一株分离自断奶仔猪小肠黏膜的肠乳杆菌L1(Lactobacillus intestinalis)体外发酵特性,及其代谢产物对病原性大肠杆菌Escherichia coli K88和O138存活的影响.体外发酵结果表明:发酵12 h后,L1菌液pH值迅速降至3.90,并产生大量乳酸,为104.08 mmol/L.L1菌株代谢产物对K88和O138体外生长抑制的动力学研究表明:L1菌株代谢产物对K88和O138存活具有很强的抑制作用;L1菌株发酵液与含相同浓度乳酸的自制培养液比较结果表明:乳酸在L1菌株代谢物对K88和O138存活抑制中发挥了主要作用;K88和O138对pH 4.5的MRS培养液具有一定的耐受能力.     

Genome sequences and phylogenetic analysis of K88- and F18-positive porcine enterotoxigenic Escherichia coli

Porcine enterotoxigenic Escherichia coli (ETEC) continues to result in major morbidity and mortality in the swine industry via postweaning diarrhea. The key virulence factors of ETEC strains, their serotypes, and their fimbrial components have been well studied. However, most studies to date have focused on plasmid-encoded traits related to colonization and toxin production, and the chromosomal backgrounds of these strains have been largely understudied. Here, we generated the genomic sequences of K88-positive and F18-positive porcine ETEC strains and examined the phylogenetic distribution of clinical porcine ETEC strains and their plasmid-associated genetic content. The genomes of porcine ETEC strains UMNK88 and UMNF18 were both found to contain remarkable plasmid complements containing known virulence factors, potential novel virulence factors, and antimicrobial resistance-associated elements. The chromosomes of these strains also possessed several unique genomic islands containing hypothetical genes with similarity to classical virulence factors, although phage-associated genomic islands dominated the accessory genomes of these strains. Phylogenetic analysis of 78 clinical isolates associated with neonatal and porcine diarrhea revealed that a limited subset of porcine ETEC lineages exist that generally contain common toxin and fimbrial profiles, with many of the isolates belonging to the ST10, ST23, and ST169 multilocus sequencing types. These lineages were generally distinct from existing human ETEC database isolates. Overall, most porcine ETEC strains appear to have emerged from a limited subset of E. coli lineages that either have an increased propensity to carry plasmid-encoded virulence factors or have the appropriate ETEC core genome required for virulence.     

The changes of catechins during the fermentation of green tea

The dynamics of tea catechins and organic acids in fermented fluid and yeast cells were studied. The concentration of eight kinds of catechins solution decreased by from 29.6% to 47.6%, respectively, some catechins were absorbed and accumulated by yeast cells, but the amount in the cells was very low during the fermentation process. The investigation of catechins resolved in four citrate buffers with a pH range of 2.6-5.6 for 18 h showed that most catechins were stable in buffer solutions of pH 4.6 and 5.6, and significant losses took place in solutions of pH 2.6 and 3.6. However, most catechins were released and recovered by adjusting the pH value to 5.6, which suggested that catechins in extremely acidic buffer solutions might reversibly combine each other or with other compounds.     

N-ethylmaleimide desensitizes pH-dependence of K+/H+ antiporter in a marine bacterium, Vibrio alginolyticus

The K+/H+ antiporter of a marine bacterium, Vibrio alginolyticus, is strongly dependent upon the cytoplasmic pH and functions only at an internal pH above 7.7. In alkaline buffer with an outwardly directed chemical gradient of K+ (delta pK), the internal pH was maintained at about 7.7. Addition of N-ethylmaleimide (NEM) released cellular K+ and acidified the cytosol below pH 7.7. The NEM effect was reversed by the addition of 2-mercaptoethanol: K+ efflux ceased, and the internal pH returned to about 7.7. In acidic buffer, the internal pH was also regulated at about 7.6 even in the absence of delta pK. Following addition of NEM, the internal pH decreased below 7.6, dissipating delta pH. These results suggest that NEM desensitizes the pH-dependence of the K+/H+ antiporter, allowing the antiporter to function at an internal pH below 7.7.