What Is the Diagnostic Accuracy of Alpha-Defensin and Leukocyte Esterase Test in Periprosthetic Shoulder Infection?

BACKGROUND: The diagnosis of periprosthetic joint infection (PJI) after total shoulder arthroplasty (TSA) is challenging, especially in patients with Cutibacterium (formerly Propionibacterium) acnes infection. Despite the increasing number of patients with PJI of the shoulder, there are still no robust data regarding diagnostic tests in detecting shoulder PJI. QUESTIONS/PURPOSES: (1) What are the sensitivity, specificity, and negative- and positive-predictive values for the alpha-defensin enzyme-linked immunosorbent assay test in detecting PJI after TSA? (2) What are the diagnostic accuracies in detecting shoulder PJI for synovial alpha-defensin, leukocyte esterase Test, and serum C-reactive protein (CRP)? METHODS: All patients with painful TSA, who underwent joint aspiration to validate or exclude a PJI, between July 2015 and February 2018 were enrolled in this single-center study. Further indications for aspiration were as follows: planned revision arthroplasty, early loosening and clinical signs of infections, especially serum CRP elevation. A total of 121 patients were aspirated to exclude or verify a PJI, and 16 patients were excluded. In all, 105 patients with a mean age of 68 years (± 12 years) were included for analysis. Patients who underwent TSA were considered aseptic or septic according to the Musculoskeletal Infection Society criteria. Twenty-four patients had a PJI, and the remaining 81 patients were in the aseptic group. The microbiologic evaluation including polymicrobial infection showed C. (formerly P.) acnes in 15 patients (63%). Synovial fluid was then analyzed using microbiology cultures, alpha-defensin immunoassay, and leukocyte esterase. The specificity, sensitivity, and positive-predictive and negative-predictive values were calculated for each test. RESULTS: The overall accuracy for alpha-defensin was 91% (95% confidence interval [CI], 84.4-96); sensitivity was 75% (95% CI, 53-90), specificity was 96% (95% CI, 90-99), negative predictive value was 93% (95% CI, 85-97), and positive predictive value was 86% (95% CI, 64-97). In contrast, the overall accuracy for leukocyte esterase was 76% (95% CI, 61-88), sensitivity was 50% (95% CI, 21-79), specificity was 87% (95% CI, 69-96), positive predictive value 60% (95% CI, 26-88) and negative predictive value was 81% (95% CI, 64-93). CONCLUSIONS: Summarizing the study results, the alpha-defensin ELISA and leukocyte esterase tests had less sensitivity in detecting shoulder PJI than previously reported TKA or THA results. The quality and low amount of joint fluid is the difficult part of the diagnostic. C. (formerly P.) acnes was the most common cause of PJI. Focusing on low-grade infections, alpha-defensin has shown its advantages in diagnosing PJI regardless pathogen virulence. Since the diagnostic of a PJI is always a synopsis of findings, the alpha-defensin and leukocyte esterase test can be used as adjunct diagnostic tool in patients with painful TSA. We propose further prospective studies to improve the diagnostic and confirm the results. LEVEL OF EVIDENCE: Level III, diagnostic study.

Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells.

Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.

Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-ß-lactamase found in a Pseudomonas aeruginosa clinical isolate.

Objectives: To characterize a novel subclass B1 metallo-ß-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate. Methods: The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat . Results: P. aeruginosa NRZ-03096 was resistant to all tested ß-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to ß-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem. Conclusions: The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .

Molecular analysis of the ramRA locus in clinical Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline.

Mutations in ramR, a negative regulator of ramA which stimulates transcription of acrA/-B encoding the multidrug efflux pump AcrAB-TolC, were recently shown to result in reduced susceptibility to tigecycline in Klebsiella pneumoniae. We analysed six non-duplicate K. pneumoniae isolates with elevated MICs to tigecycline. All isolates showed transcriptional up-regulation of ramA and acrB as demonstrated by Northern blot and quantitative real-time PCR. Sequencing of the ramR gene revealed deletions in five of the isolates and a premature stop codon in one isolate. Transformation of the wild-type ramR gene but not of any of the detected mutant ramR genes into a ramR-mutant K. pneumoniae strain restored tigecycline susceptibility and repressed ramA and acrB transcription to wild type levels. Thus, our study confirms the role of inactivating mutations in the ramR gene in tigecycline resistance.

Renal intercalated cells are rather energized by a proton than a sodium pump.

The Na(+) concentration of the intracellular milieu is very low compared with the extracellular medium. Transport of Na(+) along this gradient is used to fuel secondary transport of many solutes, and thus plays a major role for most cell functions including the control of cell volume and resting membrane potential. Because of a continuous leak, Na(+) has to be permanently removed from the intracellular milieu, a process that is thought to be exclusively mediated by the Na(+)/K(+)-ATPase in animal cells. Here, we show that intercalated cells of the mouse kidney are an exception to this general rule. By an approach combining two-photon imaging of isolated renal tubules, physiological studies, and genetically engineered animals, we demonstrate that inhibition of the H(+) vacuolar-type ATPase (V-ATPase) caused drastic cell swelling and depolarization, and also inhibited the NaCl absorption pathway that we recently discovered in intercalated cells. In contrast, pharmacological blockade of the Na(+)/K(+)-ATPase had no effects. Basolateral NaCl exit from ß-intercalated cells was independent of the Na(+)/K(+)-ATPase but critically relied on the presence of the basolateral ion transporter anion exchanger 4. We conclude that not all animal cells critically rely on the sodium pump as the unique bioenergizer, but can be replaced by the H(+) V-ATPase in renal intercalated cells. This concept is likely to apply to other animal cell types characterized by plasma membrane expression of the H(+) V-ATPase.

Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4.

An outbreak caused by Shiga-toxin­producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance.

Prevalence and genotypes of extended spectrum beta-lactamases in Enterobacteriaceae isolated from human stool and chicken meat in Hamburg, Germany.

Chicken meat has been proposed to constitute a source for extended spectrum beta-lactamase (ESBL)-carrying Enterobacteriaceae that colonize and infect humans. In this study the prevalence of ESBL-producing Enterobacteriaceae in stool samples from ambulatory patients who presented in the emergency department of the University Medical Centre Hamburg-Eppendorf with gastrointestinal complains and in chicken meat samples from the Hamburg region were analysed and compared with respect to ESBL-genotypes, sequence types and antibiotic resistance profiles. Twenty-nine (4.1%) of 707 stool samples and 72 (60%) of 120 chicken meat samples were positive for ESBL-producing Enterobacteriaceae. The distribution of ESBL genes in the stool vs. chicken meat isolates (given as % of total isolates from stool vs. chicken meat) was as follows: CTX-M-15 (38% vs. 0%), CTX-M-14 (17% vs. 6%), CTX-M-1 (17% vs. 69%), SHV-12 (3% vs. 18%) and TEM-52 (3% each). Comparison of ESBL- and multilocus sequence type revealed no correlation between isolates of human and chicken. Furthermore, ESBL-producing E. coli from stool samples were significantly more resistant to fluoroquinolones, aminoglycosides and/or trimethoprim-sulfamethoxazole than chicken isolates. The differences in ESBL-genotypes, sequence types and antibiotic resistance patterns indicate that in our clinical setting chicken meat is not a major contributor to human colonization with ESBL-carrying Enterobacteriaceae.

sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis-dependent release of eDNA.

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant-associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm-negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp- and eDNA-dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.

First case of bloodstream infection caused by Mixta hanseatica sp. nov., a novel species within the Mixta genus of the Erwiniaceae family.

Members of the Erwiniaceae family very rarely cause infections in humans. Here we describe the first case of a bloodstream infection due to Mixta hanseatica sp. nov., a novel member of the Erwiniaceae family.

Genomics of Invasive Cutibacterium acnes Isolates from Deep-Seated Infections.

Cutibacterium acnes, formerly known as Propionibacterium acnes, is a commensal of the human pilosebaceous unit but also causes deep-seated infection, especially in the context of orthopedic and neurosurgical foreign materials. Interestingly, little is known about the role of specific pathogenicity factors for infection establishment. Here, 86 infection-associated and 103 commensalism-associated isolates of C. acnes were collected from three independent microbiology laboratories. We sequenced the whole genomes of the isolates for genotyping and a genome-wide association study (GWAS). We found that C. acnes subsp. acnes IA1 was the most significant phylotype among the infection isolates (48.3% of all infection isolates; odds ratio [OR] = 1.98 for infection). Among the commensal isolates, C. acnes subsp. acnes IB was the most significant phylotype (40.8% of all commensal isolates; OR = 0.5 for infection). Interestingly, C. acnes subsp. elongatum (III) was rare overall and did not occur at all in infection. The open reading frame-based GWAS (ORF-GWAS) did not show any loci with a strong signal for infection association (no P values of ≤0.05 after adjustment for multiple testing; no logarithmic OR [logOR] of ≥|2|). We concluded that all subspecies and phylotypes of C. acnes, possibly with the exception of C. acnes subsp. elongatum, are able to cause deep-seated infection given favorable conditions, most importantly related to inserted foreign material. Genetic content appears to have a small effect on the likelihood of infection establishment, and functional studies are needed to understand the individual factors contributing to deep-seated infections caused by C. acnes. IMPORTANCE Opportunistic infections emerging from human skin microbiota are of ever-increasing importance. Cutibacterium acnes, being abundant on the human skin, may cause deep-seated infections (e.g., device-associated infections). Differentiation between invasive (i.e., clinically significant) C. acnes isolates and sole contaminants is often difficult. Identification of genetic markers associated with invasiveness not only would strengthen our knowledge related to pathogenesis but also could open ways to selectively categorize invasive and contaminating isolates in the clinical microbiology lab. We show that in contrast to other opportunistic pathogens (e.g., Staphylococcus epidermidis), invasiveness is apparently a broadly distributed ability across almost all C. acnes subspecies and phylotypes. Thus, our work strongly supports an approach in which clinical significance is judged from clinical context rather than by detecting specific genetic traits.

Staphylococcus epidermidis uses distinct mechanisms of biofilm formation to interfere with phagocytosis and activation of mouse macrophage-like cells 774A.1.

Assembly of adherent biofilms is the key mechanism involved in Staphylococcus epidermidis virulence during device-associated infections. Aside from polysaccharide intercellular adhesin (PIA), the accumulation-associated protein Aap and the extracellular matrix binding protein Embp act as intercellular adhesins, mediating S. epidermidis cell aggregation and biofilm accumulation. The aim of this study was to investigate structural features of PIA-, Aap-, and Embp-mediated S. epidermidis biofilms in more detail and to evaluate their specific contributions to biofilm-related S. epidermidis immune escape. PIA-, Embp-, and Aap-mediated biofilms exhibited substantial morphological differences. Basically, PIA synthesis induced formation of macroscopically visible, rough cell clusters, whereas Aap- and Embp-dependent biofilms preferentially displayed a smooth layer of aggregated bacteria. On the microscopic level, PIA was found to form a string-like organized extracellular matrix connecting the bacteria, while Embp produced small deposits of intercellular matrix and Aap was strictly localized to the bacterial surface. Despite marked differences, S. epidermidis strains using PIA, Aap, or Embp for biofilm formation were protected from uptake by J774A.1 macrophages, with similarly efficiencies. In addition, compared to biofilm-negative S. epidermidis strains, isogenic biofilm-forming S. epidermidis induced only a diminished inflammatory J774A.1 macrophage response, leading to significantly (88.2 to 88.7%) reduced NF-κB activation and 68.8 to 83% reduced interleukin-1ß (IL-1ß) production. Mechanical biofilm dispersal partially restored induction of NF-κB activation, although bacterial cell surfaces remained decorated with the respective intercellular adhesins. Our results demonstrate that distinct S. epidermidis biofilm morphotypes are similarly effective at protecting S. epidermidis from phagocytic uptake and at counteracting macrophage activation, providing novel insights into mechanisms that could contribute to the chronic and persistent course of biofilm-related S. epidermidis foreign material infections.

Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes Yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissue.

Pathogenic Yersinia species inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of the Y. enterocolitica serogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in the Y. enterocolitica serogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, the Yersinia mutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured, Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard, Y. enterocolitica O8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism.

Mice with targeted Slc4a10 gene disruption have small brain ventricles and show reduced neuronal excitability.

Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Slc4a10 gene, which encodes the Na(+)-coupled Cl(-)-HCO(3)(-) exchanger Slc4a10 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4a10 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4a10 also was expressed in neurons. Within the hippocampus, the Slc4a10 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridine-induced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure.

NKCC1-dependent GABAergic excitation drives synaptic network maturation during early hippocampal development.

A high intracellular chloride concentration in immature neurons leads to a depolarizing action of GABA that is thought to shape the developing neuronal network. We show that GABA-triggered depolarization and Ca2+ transients were attenuated in mice deficient for the Na-K-2Cl cotransporter NKCC1. Correlated Ca2+ transients and giant depolarizing potentials (GDPs) were drastically reduced and the maturation of the glutamatergic and GABAergic transmission in CA1 delayed. Brain morphology, synaptic density, and expression levels of certain developmental marker genes were unchanged. The expression of lynx1, a protein known to dampen network activity, was decreased. In mice deficient for the neuronal Cl(-)/HCO(3)(-) exchanger AE3, GDPs were also diminished. These data show that NKCC1-mediated Cl(-) accumulation contributes to GABAergic excitation and network activity during early postnatal development and thus facilitates the maturation of excitatory and inhibitory synapses.

Transcriptional ERRgamma2-mediated activation is regulated by sentrin-specific proteases.

Modification with SUMOs (small ubiquitin-related modifiers) has emerged as an important means of regulating the activity of transcription factors, often by repressing their activity. The ERRgamma [oestrogen receptor-related receptor gamma; ERR3 or NR3B3 (nuclear receptor subfamily 3, group B, gene3)] is a constitutively active orphan nuclear receptor. A PDSM, (phosphorylation-dependent sumoylation motif) is located in the close vicinity of the N-terminally located ERRgamma2-specific AF-1 (activation function-1). Its function can be replaced by an NDSM (negatively charged amino acid-dependent sumoylation motif). A mutational analysis reveals that ERRgamma2 activity is modulated through sumoylation of a lysine residue at position 40, which in turn is regulated by phosphorylation. Phosphorylation at the +5 position relative to the sumoylation target is directly visualized by a high-resolution EMSA (electrophoretic mobility-shift assay). Sumoylation represses the activity of ERRgamma both with and without forced expression of the PGC-1beta (peroxisome-proliferator-activated receptor gamma co-activator-1beta). Fusion proteins of a heterologous DNA-binding domain with the ERRgamma2 N-terminus demonstrate the function of the PDSM as the RF-1 (repression function-1) for the neighbouring AF-1. De-repression is achieved by co-expression of sentrin/SENP (sentrin-specific protease) family members. Together, our results demonstrate reversible phosphorylation-dependent sumoylation as a means to regulate the activity of an orphan nuclear receptor.

Nuclear factor I-A represses expression of the cell adhesion molecule L1.

BACKGROUND: The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. RESULTS: We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. CONCLUSION: Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF).

The murine AE4 promoter predominantly drives type B intercalated cell specific transcription.

AE4 is an anion exchanger almost exclusively expressed in the collecting ducts of the kidney. This very restricted expression prompted us to analyze its transcription in more detail. 5' RACE yielded alternative transcriptional start sites that are predicted to code for N-terminal protein variants. Comparison of the 5' genomic sequence between species identified a transcriptionally active region with three conserved spans. In transgenic mice beta-galactosidase expression driven by this fragment resembled endogenous AE4 expression and was predominantly restricted to type B intercalated cells. Hence this promoter could prove useful to target type B intercalated cells by genetic approaches.

Mice with a targeted disruption of the Cl-/HCO3- exchanger AE3 display a reduced seizure threshold.

Neuronal activity results in significant pH shifts in neurons, glia, and interstitial space. Several transport mechanisms are involved in the fine-tuning and regulation of extra- and intracellular pH. The sodium-independent electroneutral anion exchangers (AEs) exchange intracellular bicarbonate for extracellular chloride and thereby lower the intracellular pH. Recently, a significant association was found with the variant Ala867Asp of the anion exchanger AE3, which is predominantly expressed in brain and heart, in a large cohort of patients with idiopathic generalized epilepsy. To analyze a possible involvement of AE3 dysfunction in the pathogenesis of seizures, we generated an AE3-knockout mouse model by targeted disruption of Slc4a3. AE3-knockout mice were apparently healthy, and neither displayed gross histological and behavioral abnormalities nor spontaneous seizures or spike wave complexes in electrocorticograms. However, the seizure threshold of AE3-knockout mice exposed to bicuculline, pentylenetetrazole, or pilocarpine was reduced, and seizure-induced mortality was significantly increased compared to wild-type littermates. In the pyramidal cell layer of the hippocampal CA3 region, where AE3 is strongly expressed, disruption of AE3 abolished sodium-independent chloride-bicarbonate exchange. These findings strongly support the hypothesis that AE3 modulates seizure susceptibility and, therefore, are of significance for understanding the role of intracellular pH in epilepsy.

Tandem Affinity Purification of SBP-CBP-tagged Type Three Secretion System Effectors.

Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by co-immunoprecipitation. The tandem affinity purification (TAP) method allows an unbiased screen of potential interaction partners of bacterial effectors in a physiological approach: target cells can be infected with a bacterial strain harboring the TAP-tagged bacterial effector protein which is translocated in the host similar as under physiological infection conditions. No transfection and overexpression of the bacterial protein in the eukaryotic host are needed. Therefore, also host target cells not easy to transfect can be analyzed by this method. Moreover, the two consecutive affinity tags Calmodulin-Binding-Peptide (CBP) and Streptavidin-Binding-Peptide (SBP) fused to the translocated bacterial protein allow an outstanding clear purification of protein complexes formed between the bacterial protein of interest and host cell proteins with less occurrence of contaminants. Mass spectrometry allows an unbiased identification of interacting eukaryotic proteins.

The forkhead transcription factor Foxi1 directly activates the AE4 promoter.

Intercalated cells are highly specialized cells within the renal collecting duct epithelium and play an important role in systemic acid-base homoeostasis. Whereas type A intercalated cells secrete protons via an apically localized H+-ATPase, type B intercalated cells secrete HCO3-. Type B intercalated cells specifically express the HCO3-/Cl- exchanger AE4 (anion exchanger 4), encoded by Slc4a9. Mice with a targeted disruption of the gene for the forkhead transcription factor Foxi1 display renal tubular acidosis due to an intercalated cell-differentiation defect. Collecting duct cells in these mice are characterized by the absence of inter-calated cell markers including AE4. To test whether Slc4a9 is a direct target gene of Foxi1, an AE4 promoter construct was generated for a cell-based reporter gene assay. Co-transfection with the Foxi1 cDNA resulted in an approx. 100-fold activation of the AE4 promoter construct. By truncating the AE4 promoter at the 5'-end, we demonstrate that a fragment of approx. 462 bp upstream of the transcription start point is sufficient to mediate activation by Foxi1. Sequence analysis of this region revealed at least eight potential binding sites for Foxi1 in both sense and antisense orientation. Only one element was bound by recombinant Foxi1 protein in bandshift assays. Mutation of this site abolished both binding in bandshift assays and transcriptional activation by co-transfection of Foxi1 in the reporter gene assay. We thus identify the AE4 promoter as a direct target of Foxi1.