The aim was to assess the attainability and outcome of the lupus low disease activity state (LLDAS) in the early stages of systemic lupus erythematosus (SLE).LLDAS prevalence was evaluated at 6 (T1) and 18 (T2) months after diagnosis and treatment initiation (T0) in a monocentric cohort of 107 (median disease duration 9.7 months) prospectively followed Caucasian patients with SLE. Reasons for failure to achieve LLDAS were also investigated. Multivariate models were built to identify factors associated with lack of LLDAS achievement and to investigate the relationship between LLDAS and Systemic Lupus International Collaboration Clinics (SLICC)/Damage Index (SDI) accrual.p = 0.019) and T2 . Presence of any organ damage (SDI ≥1) at T2 was significantly associated with lack of LLDAS at T1  and older age at diagnosis .There were 47 (43.9%) patients in LLDAS at T1 and 48 (44.9%) at T2. The most frequent unmet LLDAS criterion was prednisolone dose >7.5 mg/day (83% of patients with no LLDAS at T1). Disease manifestations with the lowest remission rate during follow up were increased anti-double-stranded DNA , low serum complement fractions (73.2% and 66.3%) and renal abnormalities (46.4% and 28.6%). Renal involvement at T0 was significantly associated with failure to achieve LLDAS both at T1 (OR 7.8, 95% CI 1.4–43.4; LLDAS is a promising treatment target in the early stages of SLE, being attainable and negatively associated with damage accrual, but it fit poorly to patients with renal involvement. Systemic lupus erythematosus (SLE) is a chronic multisystem disease, characterized by wide heterogeneity in clinical presentation, course and responsiveness to therapy , 2. DespThe treat-to-target (T2T) approach, successfully applied in rheumatoid arthritis and other non-rheumatic conditions, is a therapeutic strategy aiming to improve disease outcomes through the achievement of a pre-specified goal . An inteEarly damage was demonstrated to be associated with lower 10-year survival, such that patients with initial damage have a fourfold higher mortality rate compared to those with no early damage . In ordeThis study primarily aimed to assess the frequency of LLDAS achievement and its association with early damage accrual in a homogeneous cohort of Caucasian patients with SLE prospectively assessed during the first 18 months of treatment after diagnosis. The secondary aim was to identify the main reasons for failure to achieve LLDAS.Data from patients included in the Cagliari  SLE cohort  between Retrospective analysis of prospectively collected data was performed, reviewing medical records and clinical files. Baseline was fixed at the time of diagnosis, which corresponded to treatment initiation (T0). No specific recommendations are currently available on the appropriate time to assess the target achievement in a T2T strategy for SLE . In ordeDemographic factors including gender, age at onset and diagnosis were collected. Disease onset was defined as the time of appearance of the first clinical classification criterion. Disease duration was the time period between disease onset and diagnosis. The number of 1997 ACR criteria presented from disease onset up to the baseline was recorded. At baseline, antinuclear antibodies (ANA) (tested by indirect immunofluorescence (IIF), using Hep2 cell substrate, positivity was defined as a titre >1:160), anti-Ro/SSA, anti-La/SSB, anti-Sm, anti-RNP, lupus anticoagulant, anticardiolipin  and anti-B2-glicoprotein1  antibodies were assessed. Anti-dsDNA antibodies  and C3 (nv 90–180 mg/dL), C4 (nv 10–40 mg/dL) serum complement fractions were quarterly tested.At each visit, disease activity was assessed using the SLEDAI-2K score and the PGA (0–3) . Damage t test or Mann–Whitney test for quantitative variables, and the Chi-square test or Fisher's exact test for qualitative variables. Variables with a p value <0.1 on univariate analysis were included in a multivariate model for stepwise logistic regression. The odds ratio (OR) with 95% confidence interval (95% CI) was calculated.Normally and non-normally distributed variables were summarized using mean ± standard deviation (SD) and median with interquartile range (IQR), respectively. Univariate and multivariate analysis were performed to evaluate potential factors associated with failure to achieve LLDAS. Univariate analysis was performed using the two-sample Student p value <0.05. MedCalc® statistical software  was used.To assess the association between lack of LLDAS achievement and damage development, a logistic regression model was created. Occurrence of any damage (SDI ≥1) at 18 months was included as the dependent variable, whereas age, disease duration, male gender, average daily steroids dosage, renal involvement, use of anti-malarial or immunosuppressant drugs  and failOverall, 178 new patients joined the Cagliari  SLE cohort during the study interval. The study cohort consisted of 107 (60.1%). The relevant features of Caucasian patients with SLE who fulfilled the inclusion criteria for study enrolment are summarized in Table At T1 (6 months), LLDAS was achieved by 47 (43.9%) patients. Focusing on unmet criteria for LLDAS in the remaining 60 (56.1%) patients: 50 (83.3%) were not on prednisolone ≤7.5 mg daily and 29 of them had SLEDAI-2K ≤4 and PGA ≤1; 7 (11.7%) did not have SLEDAI-2K ≤4 or PGA ≤1 with prednisolone dosage ≤7.5 mg/day, 3 (5.0%) experienced new manifestation , 48 (44.9%) patients were in LLDAS; 33 of them achieved LLDAS at T1 and were still in this condition at T2, whereas 15 reached LLDAS within the interval between T1 and T2 for the first time. Out of 59 (55.1%) patients who were not in LLDAS at T2, 45 had never been in LLDAS and 14 were in LLDAS at T1 but no longer at T2 ), higher SLEDAI-2K score (median 13.0 (9.0–18.0) vs 8 (7.2–10.0); p < 0.001)), positive (>10UI/mL) anti-dsDNA antibodies (49 (81.7%) vs 29 (61.7%); p = 0.013), lower serum C3 (median 65.5 (40.1–80.5) vs 82.0 (67.0–90.8) mg/dL; p = 0.002) and C4 values (10.0 (4.0–13.0) vs 13.0 (10.0–16.0) mg/dL; p = 0.013), higher prednisolone dose (median 25 (14–38) vs 8 (5–15) mg/day; p < 0.001) and immunosuppressant drug use (45 (75.0%) vs 23 (48.9%); p = 0.010). On multivariate analysis renal involvement  and C4 level  were confirmed to be associated with failure to achieve LLDAS  vs 2 (4.3%); p = 0.016), discoid rash (7 (11.9%) vs 0; p = 0.007), any aPLs (10 (16.9%) vs 16 (33.3%); p = 0.016) and average prednisolone dosage (median 12.5 (6.7–21.6) vs 8.0 (5.0–14.4) mg/day; p = 0.0159) were associated with no LLDAS achievement at 18 months. On multivariate analysis, renal involvement was the only factor independently associated with failure to achieve LLDAS at T2 .Demographic, clinical and serological variables at baseline were also analysed as potential predictors of lack of LLADS at T2. On univariate analysis renal involvement (21 (36%) vs 6 (13%); After 18 months since diagnosis (T2), 23 patients out of 107 (21.5%) had SDI≥1 (range 0-3). Overall, 27 SDI items of damage were recorded in 23 patients at T2: 9 were definitely steroid-related , 8 possibly steroid-related  and 9 independent of corticosteroid use .p = 0.009) and age at diagnosis  were independently associated with the presence of any damage at T2  had not achieved LLDAS at T1 and 18 (78.3%) at T2. In the logistic regression model failure to achieve LLDAS at T1 . In the study by Golder et al., prednisolone dosage was an unmet criterion in 57% of patients who did not achieve LLDAS . In a stp = 0.019) and 18 months  from treatment initiation. In a cross-sectional study, increased anti-dsDNA , renal involvement , low complement  shorter disease duration  and history of discoid rash  were identified as negatively associated with LLDAS [Renal involvement and serological disorders, consisting of low complement and increased anti-dsDNA, had the lowest remission rate in our cohort. However, renal involvement at baseline was the most important factor associated with failure to achieve LLDAS at 6 months (OR 7.8, 95% CI 1.4–43.4; th LLDAS . These fWhether or not serological disorders should be considered in the definition of minimal disease activity it is a matter of debate . PersistThis study has some limitations. First, the relatively small sample size may have hampered the study results. Nevertheless, by enrolling consecutively diagnosed patients at the time of treatment initiation and following them up prospectively, we added some novel information on LLDAS as a potential treatment target. Second, the retrospective design of the study prevented us from testing LLDAS criterion validity by comparing it with other treatment targets such as the SLE Responder Index .In conclusion, LLDAS is a promising treatment target in SLE, being attainable and negatively associated with damage accrual in the early stages of disease. However, it seems to poorly fit with the heterogeneity of clinical presentation in patients with SLE, mostly in those with renal involvement. On the one hand, the poor LLDAS attainability in patients with renal involvement could be due to greater severity and lasting disease activity in the renal domain. On the other hand, it could be due to the unsuitability of LLDAS in capturing the minimal disease activity in patients with renal involvement. Finally, greater consensus should be reached on the definition of serological disorders and prednisolone dose to be included in a unique definition of minimally acceptable disease activity.


An. funestus is increasingly reported across Africa, threatening the sustainability of pyrethroid-based control interventions, including long lasting insecticidal nets (LLINs). Managing this problem requires understanding of the molecular basis of the resistance from different regions of the continent, to establish whether it is being driven by a single or independent selective events. Here, using a genome-wide transcription profiling of pyrethroid resistant populations from southern , East (Uganda), and West Africa (Benin), we investigated the molecular basis of resistance, revealing strong differences between the different African regions. The duplicated cytochrome P450 genes (CYP6P9a and CYP6P9b) which were highly overexpressed in southern Africa are not the most upregulated in other regions, where other genes are more overexpressed, including GSTe2 in West (Benin) and CYP9K1 in East (Uganda). The lack of directional selection on both CYP6P9a and CYP6P9b in Uganda in contrast to southern Africa further supports the limited role of these genes outside southern Africa. However, other genes such as the P450 CYP9J11 are commonly overexpressed in all countries across Africa. Here, CYP9J11 is functionally characterized and shown to confer resistance to pyrethroids and moderate cross-resistance to carbamates (bendiocarb). The consistent overexpression of GSTe2 in Benin is coupled with a role of allelic variation at this gene as GAL4-UAS transgenic expression in Drosophila flies showed that the resistant 119F allele is highly efficient in conferring both DDT and permethrin resistance than the L119. The heterogeneity in the molecular basis of resistance and cross-resistance to insecticides in An. funestus populations throughout sub-Saharan African should be taken into account in designing resistance management strategies.Pyrethroid resistance in malaria vector,  Anopheles funestus s.s. is one of the major malaria vectors in Sub-Saharan Africa and is widely distributed across the continent . As in other malaria vectors, pyrethroid resistance in An. funestus has increasingly been reported in Sub-Saharan Africa from different regions, including southern  w[*]; P(Act5C-GAL4-w)E1/CyO,”“1;2”]  with homozygote UAS-CYP9J11 males. Similarly, adult F1 control progeny (control group) with the same genetic background as the experimental group but without expression of CYP9J11 were obtained by crossing virgin females from the driver strain Act5C-GAL4 and UAS recipient line males (which do not carry the pUASattb-CYP9J11 insertion).Briefly, the same predominant clone used for transgenic expression was selected to construct transgenic flies, and cloned into the pUASattB vector using primers containing restriction sites for 1Drosophila melanogaster females were performed as previously described  in 2010 as previously described .P < 0.05) for the R-S (7346) and C-S (7479) comparisons  (R-C) and to the fully susceptible laboratory strain FANG (R-S). The control mosquitoes were also compared to the susceptible FANG strain (C-S). High numbers of probes were significantly differentially expressed .The cytochrome P450 Anopheles , which was the most overexpressed with FC70.1 in R-S and 24 in C-S. This P450 has previously been shown to metabolize pyrethroids (CYP9K1 (Afun007769) with a higher fold change (FC21.5) in R-S than previously observed in southern Africa, suggesting a higher role played by this gene in Uganda. The CYP307A1 (Afun015331) exhibited a high FC in R-S (FC16.6). Other cytochrome P450s included CYP6 subfamily genes such as CYP6Z1, CYP6P5, CYP6P4, CYP6Z3, and, notably, the CYP6M8 (Afun006930), of which the ortholog from An. gambiae, CYP6M2 is responsible for pyrethroid resistance in this species (An. funestus. A particular transcript (Afun07369) had a close hit to CYP6P9a, but none of the common probes for this gene highly overexpressed in southern Africa was observed in Uganda. CYP4 subfamily genes overexpressed included CYP4H17, CYP4C36, and CYP4K2, whereas CYP9J11, from the CYP9 subfamily, was also overexpressed in both comparisons. Glutathione S-transferases (GSTs) were also significantly overexpressed in pyrethroid resistant mosquitoes from Uganda compared to the susceptible FANG strain, notably genes of the epsilon class, including GSTe1 (Afun013481) , GSTe5 (Afun009866) and GSTe2 (Afun000045), which, with FC of 2.9 and 2.1, exhibits a lower FC than the level observed in West Africa (GSTD3 (Afun008354) (FC6.6 and 5.1) and GSTD1-5 (Afun007499). Other overexpressed detoxification gene families included sulfotransferases (with Afun013870 having a high FC of 11.9 and 14.8), carboxylesterases, aldehyde oxidases, ABC transporters, and other genes commonly associated with metabolic resistance to pyrethroid (Among the most overexpressed genes commonly observed in R-S and C-S were proteases such as a trypsin-related protease (Afun008293), which was the top upregulated with FC187.8 in R-S and 83.07 in C-S. Other highly overexpressed proteases included chymotrypsin 1 (Afun013921), with FC64.4 in R-S and 49.5 in C-S. High overexpression of proteases is commonly reported in resistant mosquitoes either rethroid . CuticulA similar approach was used in Benin using the 4 × 44k chip, as was carried out before the design of the 8 × 60k. High numbers of probes were significantly differentially expressed for the R-S (5617) and C-S (7735) comparisons , most liGSTe2 (Combined_c920) was the only detoxification gene commonly overexpressed in R-C, R-S, and C-S . Two probes of the CYP9J11 were also upregulated, but between R-C and R-S only (CYP6Z1 (three probes), CYP6Z3, CYP6P1, and another probe for CYP6P9a. It also included two ABC transporter genes , an aldehyde dehydrogenase, and cuticular protein genes . Similar results were obtained for other comparisons. The lack of GO terms associated with detoxification is similar to previous studies with this microarray chip in An. funestus . Another P450, CYP9K1, although commonly overexpressed in all three countries, was significantly more highly present in Uganda (FC16.1) than in Malawi (FC2.4) and Benin (FC6.2), suggesting a bigger role for this gene in Uganda. Other commonly expressed detoxification genes included an aldehyde oxidase (AGAP006220) and a UDP glucuronosyl transferase (AGAP006222).Among the genes the most upregulated in all three regions were a trypsin-related protease gene (Afun008293), the P450 n Malawi . Among tGSTe1 and GSTd3 were common to both countries, as were the P450s CYP307A, CYP314A1, and CYP315A. For those overexpressed only in Uganda and Malawi, the P450 CYP4C27 was detected, although with a higher expression in Uganda (FC10.3) than in Malawi (FC2.1). The CYP4C36 was also upregulated in both countries, similar to GSTd1-5. Other genes are also listed in Table S2 in File S1. The list of genes overexpressed only in Malawi and Benin is dominated by the CYP6P9a and CYP6P9b with several probes, but with a far higher overexpression in Malawi for both genes , suggesting that both genes are mainly driving resistance in southern Africa.Analysis of the list of genes commonly overexpressed in only two regions revealed that, for Uganda and Benin, the GSTs GSTe2, which in Benin and Malawi was detected by probes against Combined_c920 transcript, whereas in Uganda and Malawi it was through probes for Afun000045 transcript, showing the impact of sequence polymorphism in the microarray results.Some genes common to all countries were detected through different probes, such as CYP6P9a, CYP6P9b, and GSTe2) or commonly overexpressed in all three countries (CYP9J11) were further validated by qRT-PCR. Analysis of the expression patterns confirmed the differences observed with microarrays, as both CYP6P9a and CYP6P9b were highly overexpressed only in Malawi, but just barely in Uganda and Benin, as seen with microarray , but only very low-level expression of this gene was observed in Uganda and Malawi. The common overexpression of the CYP9J11 was also validated in all three countries, although at a lower fold-change compared to the other genes.Key genes exhibiting striking differences between regions  commonly overexpressed in the three geographical regions are located in the chromosomal regions spanning the three QTL  previously detected for pyrethroid resistance in CYP9J11 full-length cDNA (1644 bp) for five clones each from Malawi and Zambia, and four from Uganda revealed a high polymorphism of this gene, with an average of 93 polymorphic sites observed for all combined 14 sequences and 17 amino acid changes observed in total (Table S3 in File S1). No evidence of directional selection was detected on CYP9J11, as shown by the lack of significant Tajima D and Fu and Li D’ estimates. No specific clades per the location of origin was observed between haplotypes, although the genetic distance tree revealed a closer genetic similarity between Malawi and Zambia than Uganda, as expected from geographical distance  compared with previous estimates reported for CYP6M7 (0.15 ± 0.0 nmol/mg protein), and for CYP6P9a (0.42–1.0 nmol/mg) and CYP6P9b (0.35–0.42 nmol/mg), respectively.5 and NADPH regeneration system. CYP9J11 metabolized permethrin and deltamethrin with significant depletions of 88.05% ± 3.23 (P < 0.0001) and 95.05% + 0.74 (P < 0.0001), respectively  than previously reported for CYP6Z1   . Initial < 0.05)  and suchKcat) and KM obtained with permethrin was 9.260 min−1 ± 1.048 and 14.39 µM ± 3.12 leading to a catalytic efficiency of 0.643 min−1 µM−1 ± 0.157. The turnover for deltamethrin (4.338 min−1 ± 1.381) was, on average, half the value obtained with permethrin, but the affinity of CYP9J11 toward deltamethrin was surprising higher (KM of 7.957 ± 1.31). The catalytic efficiency of CYP9J11 for deltamethrin was calculated as 0.545 min−1 µM−1 ± 0.195, lower than obtained with permethrin. The catalytic efficiency of this enzyme toward permethrin is higher than obtained from An. funestus pyrethroid metabolizers CYP6P9a and CYP6P9b  as described to be the case of some P450s  and low maximal catalytic rate  for bendiocarb were calculated as 0.04 min−1 ± 0.005 and 0.75 µM ± 0.2, respectively, leading to a very low catalytic efficiency of 0.053 min−1 µM−1 ± 0.0157, 12 times lower than compared with the values obtained with permethrin.me P450s . CYP9J11tic rate . The dostic rate . The VmaCYP9J11 overtranscription alone can confer pyrethroid resistance, transgenic D. melanogaster individuals were generated expressing CYP9J11 (derived from permethrin resistant field mosquitoes from Uganda) under the control of the ubiquitous Act5C-GAL4 driver. Contact bioassays performed with 2% permethrin (type I pyrethroid) and 0.15% deltamethrin (type II) revealed that CYP9J11 overtranscription alone is sufficient to confer resistance to this class of insecticide. For deltamethrin, the flies overexpressing CYP9J11 were resistant with a significantly reduced mortality/knockdown rate compared to that observed for control flies  at 1 hr, 9.5 ± 1.7% vs. 74.7 ± 6.32% (P < 0.001) at 2 hr and 56.03 ± 4.6% vs. 98.3 ± 3.3% (P < 0.001) at 24 hr . However, similar mortality rates were recorded for both experimental and control samples at the rest of the exposure times, with no significant differences observed   suggests that even overexpression of the susceptible allele provide resistance against DDT in flies, but at a significantly lower level than with the 119F resistance allele. Bioassays with permethrin revealed that only flies expressing the resistant 119F allele had significantly lower mortality rate compared to control flies not expressing GSTe2  (1 progeny of the GAL4/UAS crosses by qRT-PCR (Due to the consistent overexpression of < 0.001) . Howeverre time) . Flies e qRT-PCR . CYP6P9a and CYP6P9b was performed between permethrin-resistant and -susceptible mosquitoes from Uganda and those from Malawi. The aim was to assess whether the low expression of these genes in Uganda correlated with a higher genetic diversity of both genes in contrast to southern African, where a high overexpression was associated with a directional selection with reduced genetic diversity , haplotypes , and estimates of genetic diversity or number of nonsynonymous substitutions (Table S3 in File S1). This higher genetic diversity of both genes in Uganda correlates with their low overexpression and support a lower role of both genes in Uganda. However, the maximum likelihood trees of haplotypes of both genes for Uganda and Malawi ] in the promoter region characteristic of southern African resistant haplotypes  QTL had previously been detected, suggesting that it could be the main gene behind rp3  suggested that this gene could be involved in permethrin resistance. The significant lower mortality observed in transgenic Drosophila flies expressing the resistant 119F allele compared to those expressing the susceptible L119 allele supports the key role that allelic variation in this gene plays beside its overtranscription. As previously shown for DDT resistance, it is likely that the 119F also enlarging the GSTe2 binding cavity to facilitate access of pyrethroid, and allow either sequestration as suggested for GST action on pyrethroids (Drosophila flies to establish the phenotypic impact of the allelic variation of GSTe2 due to a single amino acid change highlights the robustness of this approach in functionally characterizing the role of candidate resistance genes in conferring resistance to insecticide. This shows that experimental results from transgenic Drosophila are very relevant to the phenotype obtained in mosquitoes, while providing the advantage that studies in Drosophila could be easily scaled up to hundreds of genes, with less work, cost and space required for storage of transgenic lines. Allelic variation was also recently shown to play an important role in the pyrethroid resistance conferred by the duplicated P450s CYP6P9a and CYP6P9b in southern African populations of An. funestus (The significant overexpression of An. funestus populations, there are significant special variations on the main genes associated with it, which could impact patterns of cross-resistance and resistance management strategies. The impact of many genes conferring resistance and cross-resistance to multiple resistant populations of An. funestus in sub-Saharan African is a challenge to resistance management. This phenomenon makes the resistance highly heterogeneous and complex, making the design of appropriate diagnostic tools operationally challenging. There is an overwhelming need for newer classes of insecticides that are safe but potent enough to control mosquito vectors of malaria and other diseases effectively. But caution must be exercised because of the presence of a number of detoxification enzymes that can confer cross-resistance, and a new insecticide may already be doomed before being deployed if resistance genes can already metabolize it.The comparative transcription analysis performed in this study between various African regions highlights that, although metabolic resistance is the common driving mechanism of pyrethroid resistance in www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.040147/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.


SLC4 family. A novel conserved Zn2+-binding motif present in the N-terminal domain of NDCBE is identified and characterized in vitro. Cellular studies confirm the Zn2+ dependent transport of two electroneutral bicarbonate transporters, NCBE and NBCn1. The Zn2+ site is mapped to a cluster of histidines close to the conserved ETARWLKFEE motif and likely plays a role in the regulation of this important motif. The combined structural and bioinformatics analysis provides a model that predicts with additional confidence the physiologically relevant interface between the cytoplasmic domain and the transmembrane domain.The sodium-driven chloride/bicarbonate exchanger (NDCBE) is essential for maintaining homeostatic pH in neurons. The crystal structure at 2.8 Å resolution of the regulatory N-terminal domain of human NDCBE represents the first crystal structure of an electroneutral sodium-bicarbonate cotransporter. The crystal structure forms an equivalent dimeric interface as observed for the cytoplasmic domain of Band 3, and thus establishes that the consensus motif VTVLP is the key minimal dimerization motif. The VTVLP motif is highly conserved and likely to be the physiologically relevant interface for all other members of the  The sodium-driven chloride/bicarbonate exchanger (NDCBE) is a member of the sodium-bicarbonate cotransporters (NBCs) within the solute carrier 4 (SLC4) gene family2. The NBCs include, among others, the electroneutral sodium-bicarbonate cotransporter (NCBE/NBCn2), which is mainly expressed in the brain especially in the choroid plexus3, and the electrogenic sodium-bicarbonate cotransporter 2 (NBCe2), which is mainly expressed in the kidney, liver and the choroid plexus4. NDCBE is electroneutral and believed to exchange one Cl− for two HCO3− and one Na+ 5. In the brain, NDCBE is primarily associated with the presynaptic vesicles of glutamatergic and GABAergic neurons6. Even though no human disease has been associated with NDCBE mutations, knockout mice exhibit defective regulation of Na+ reabsorption and reduced neuronal excitability8. In addition, low NDCBE expression levels have been reported in mouse brains in connection with chronic continuous hypoxia9. The primary structure of SLC4 transporters is divided into three major domains, an N-terminal regulatory domain, a transmembrane domain containing 14 membrane-spanning helices, and a C-terminal domain often associated with binding to carbonic anhydrase11. The transmembrane domain is largely conserved across SLC4 family members and shares more than 38% sequence identity with anion exchangers (AEs) and NBCs2. In the plasma membrane, most SLC4 transporters are believed to form functional dimers. The molecular basis for the dimerization was recently confirmed by a crystal structure of the transmembrane dimer of AE1 (PDB ID: 4YZF) reported by Arakawa et al. at 3.5 Å resolution10. The crystal structure did not include the cytoplasmic regulatory domains, and the organization of these with respect to the transmembrane dimer is still based on models. The primary structure of the isolated cytoplasmic regulatory domains shares lower sequence identity across the SLC4 family, than the transmembrane regions. Based on sequences with either low or high sequence variability, the cytoplasmic domains have been divided into regions referred to as constant regions (CRs) and variable regions (VRs)12. The VRs mainly include the different functional and regulatory motifs of SLC4 family members and their isoforms13. In addition, the VRs have been classified as intrinsically disordered regions, which are a specific dynamic type of element that lack any ordered secondary structures such as helices or strands15. The crystal structure of the cytoplasmic domain of Band 3 (cdb3)  contains a dimer interface connected by a domain-swapped β-sheet that extends into a helical segment often referred to as the ‘dimerization arm’16. The monomeric cytoplasmic domain, excluding the VR1, reveals a cytoplasmic N-terminal core domain consisting of the intertwined secondary structure elements CR1 and CR2. The intrinsically disordered region (VR2) forms a connecting linker between CR1 and CR216. A more recent structural model of the cdb3 lacking residues 1 to 55 has been determined at 2.2 Å resolution and crystallized under near physiological conditions (pH 6.5) (PDB ID: 4KY9). Structural comparison with the previous model at pH 4.5 shows only minor molecular differences mainly present on the domain surface17. Transport regulation by the SLC4 HCO3− transporters exerted by the N-terminal domain has been described previously2. However only a few publications, so far, describe that divalent metal ions like Mg2+ and Zn2+ can modulate the transport activity20. The electrogenic activity of NBCe1 was inhibited by Mg2+ (Ki of 0.01 mM) in whole-cell patch-clamp studies18, and the transport activity by AE1 was predicted to be stimulated by intracellular levels of Mg2+ 19. AE2 show partial inhibition by extracellular Zn2+ when expressed in Xenopus oocytes20 while equivalent concentration of extracellular Zn2+ had no effect on AE1 activity20. Here, we present the crystal structure of the truncated  N-terminal cytoplasmic domain from human NDCBE-D (ntcNDCBE) at 2.8 Å resolution. Structural analysis confirms the dimerization interface of previous structural models and determines that the key dimerization motif is a minimal beta-sheet that lacks the residues equivalent to the dimerization arm in cdb3. We identify and describe a novel Zn2+-binding site and the completely conserved HHH motif also found in the electroneutral sodium-bicarbonate transporters (NBCn1 and NCBE) surrounded by a shell of negatively charged residues on the surface of ntcNDCBE. Zn2+-binding properties are verified in vitro for ntcNDCBE and, combined with cellular assays performed on NCBE and NBCn1, this indicates a general Zn2+ dependence that could expand to all members of the SLC4 superfamily.The regulation and maintenance of intra- and extracellular pH homeostasis is fundamental for life. The main buffering system in humans is based on the equilibrium between HCOet al.16, the dimerization of ntcNDCBE is only dependent on the backbone interactions of the four-membered β-sheet and the possible additional hydrophobic interactions between Leu108, Leu110, Phe114, Val337, Leu340 and Val342 from each monomer. The initial dimerization interface proposed for cdb3 included an α-helix – located downstream of β10, residues 327 to 348 in AE1 (PDB ID: 1HYN) – as the dimerization arm, which was claimed to be important for dimerization16. The buried area between the 1HYN subunits is of 2500 Å, while ntcNDCBE has a buried area of 870 Å . However, the molecular interactions of these α-helices are not symmetric, as one would expect for a dimer interface21; we therefore propose, based on the present study, that the dimerization arm only plays a minor role in stabilizing the dimer interface  forms a dimer in the asymmetric unit. Each monomeric ntcNDCBE forms an α-β sandwich fold by nine α-helices and ten β-strands Fig. . The mai22. This arrangement would naturally lead to a high local concentration of single cytoplasmic domains. High local concentrations also occur during crystallization. However, to establish whether ntcNDCBE forms a dimer in solution at lower concentrations, the construct was subjected to size-exclusion chromatography (SEC), and eluted at volumes corresponding to the hydrodynamic volume of a dimer  share an overall similar fold . However, dimers of these two structures superimpose poorly despite the equivalent dimer interface, although they clearly show a pivotal movement between each monomer. By superimposing the central β-sheet  of the individual monomers of cdb3 (chain P) and ntcNDCBE (chain A), a pivotal movement of ~23° is observed between each monomer, while the central dimerization domain-swapped β-sheet  remains intact. This pivotal freedom hints that dynamic flexibility is allowed between the two monomers .The monomeric structures of ntcNDCBE and cdb3  are a specific class of bacterial enzymes that belong to the nitrogen-metabolic phosphotransferase system and have high structural homology with cdb32, particularly E. coli IIANtr . These proteins function as cytosolic regulators of membrane K+ and sugar transporters24. The fold of the cytoplasmic domain in the SLC4 family is unique; however, it does show some structural homology to IIANtr, as described previously2. The lowest RMSD score from the structural alignment was achieved by aligning all the residues of IIANtr with CR2 (α7-9 and β7-9) from ntcNDCBE . Each injection produced an exothermic reaction that decreased in magnitude with subsequent injections. The affinity of ntcNDCBE for Zn2+ was determined to originate from three equivalent binding sites, with an approximate KD of 2.3 µM and experimental thermodynamic parameters ΔH = −6130 ± 110.4 cal/mol and ΔS = 5.24 cal/mol/deg  motif, which has also been found in other Zn2+-binding proteins25, was found surface-exposed on the structure of the cytoplasmic domain of NDCBE  increase in tryptophan fluorescence as a function of Zn2+ concentration  and a double mutant . A strong reduction in tryptophan fluorescence yield was observed for all mutants  are protonated and thus unable to form a complex with any metal ions. Attempts to increase the pH after the crystals had formed or fast soak high concentrations of ZnCBE Fig. . In the CBE Fig. . The HXHCBE Fig. . Sequencily Fig. , whereasily Fig.  and thusion Fig. . To confine Fig. . The sigine Fig. . To exclnce Fig. , the incnts Fig. , suggest167 Fig. , which ives Fig.  show tha2+ on NBC activity on intracellular pH regulation in mammalian cells two setups were investigated. First, the effect of Zn2+ on isolated slc4a10 activity was determined in NIH-3T3 fibroblasts stably transfected with slc4a10. The Na+ and HCO3− dependent NBC activity (dpHi/dt) in the cell line was reduced by 70% in the presence of 10 µM ZnCl2 (n = 5 (control) and n = 4 (ZnCl2), p = 0.0253, unpaired t-test, Fig. slc4a10 activity. Furthermore, the pH recovery in isolated rat mesenteric small arteries  was 0.019 ± 0.002 sec−1 (n = 5) and 0.013 ± 0.001 sec−1 (n = 5), in the absence and presence of Zn2+, respectively .To investigate the physiological effect of ZnSLC4 member is available. The recent crystal structure of the AE1 transmembrane domain only allows speculation with regard to which surface of the cytoplasmic domain dimer face the transmembrane dimer. It is common for most proteins that the core is higher conserved compared to the surface-exposed regions27. The conserved surface residues that form structural patches hint at functional importance. Earlier in this paper, we presented evidence that the dimer interface found in the cdb3 dimer is also present in the here-described ntcNDCBE dimer. The presented structural evidence thus indicates that the dimer interface is indeed physiologically relevant. The dimers all form a two-fold rotational axis that is perpendicular to the β10 A/β10B sheet, leaving only two surfaces in the dimer with conserved residues facing the same direction, as described for typical homodimeric proteins such as superoxide dismutase and interleukin 1028. In ntcNDCBE, one of these surfaces clearly displays patches of higher conservation compared with the other  has a predicted affinity in the low micromolar range37. This affinity is in the same range as the identified Zn2+-binding site of ntcNDCBE, which suggests the possibility of a specific role for the observed interaction between Zn2+ and the N-terminal cytoplasmic domain of NDCBE in presynaptic vesicles. The impact of Zn2+ on NCBE activity suggests that the effect of Zn2+ binding is to reduce NBC activity. This interpretation was supported by the finding of an inhibitory effect of 10 µM Zn2+ on recovery from acidosis in smooth muscle cells of rat mesenteric small arteries most likely mediated by an inhibitory effect on NBCn1.Variations in intracellular Zn2+ binding to the regulatory domain of NBC’s suggests an overall regulatory role of Zn2+ on membrane transport of acid/base equivalents. In the case of NDCBE, the Zn2+ binding may aid in the ion transfer across the presynaptic membrane and thus play a role in neuronal signalling. The inherent pH dependence of this Zn2+ site may also reflect the site of pH sensitivity present in the NBCs. As small concentrational variations in intracellular Zn2+ would affect the equilibrium between the histidine site and free Zn2+ ions, and this would consequentely act as a pH sensor.In conclusion, Zn21, we propose the most likely orientation of this domain in relation to the transmembrane domain. We propose that the interface of the N-terminal cytoplasmic domain that interacts with the transmembrane domain is on the opposite side of the dimer compared with that suggested by Gill et al. for ntcNBCe1-A38. This previous suggestion was based on the localization of three lysine and three leucine residues. Of these six residues from NBCe1-A, only Leu152 and Lys157 (as arginine) are conserved. The N-terminal domain residues responsible for the interaction will most likely not interact with the membrane itself but with residues in the transmembrane domain. In the proposed model of NDCBE  and the complete N-terminal core domain (amino acids 40–425) of human NDCBE (SLC4A8) isoform D (uniprot ID: Q2Y0W8-4) were purchased (GenScript). The Quikchange Lightning Site-Directed Mutagenesis kit (Agilent) was used to mutate the ntcNDCBE constructs used in this study, yielding ntcNDCBE (amino acids 40–344), ntcNDCBE_H167A, ntcNDCBE_H169A, ntcNDCBE_H167A, H169A and ntcNDCBE_W80F. The ntcNCBE and ntcNDCBE constructs were N-terminally tagged with a hexa-histidine tag and TEV protease cleavage site, which were present in the vector. E. coli BL21 Gold (Agilent) was used as the expression strain for ntcNDCBE and ntcNCBE constructs. The cells were grown by fermentation in terrific broth (TB) medium supplemented with 25 µg/mL kanamycin to an OD600 of 2.0 at 37 °C. Protein expression was induced with 0.3 mM IPTG at 18 °C O/N. The cells were harvested by centrifugation at 48,000 × g. All subsequent steps were carried out at 4 °C. Cells were lysed in buffer A  and 1 µg/mL DNase I) using an Emulsiflex high-pressure homogenizer (Avestin). The lysate was cleared by centrifugation at 48,000 × g for 20 min and applied to a HisTrap HP column  pre-equilibrated in buffer B . The protein was eluted with buffer C (buffer B supplemented with 300 mM imidazole) and dialyzed overnight against buffer B in the presence of TEV protease  against buffer B to cleave the his-tag. The cleaved construct was loaded onto an IMAC column pre-equilibrated in buffer B. The flowthrough fractions were concentrated to 10 mg/mL  and subjected to SEC on a Superdex S200 column  equilibrated with buffer D . The peak fractions were pooled and concentrated to 10 mg/mL. The constructs were treated with EDTA  and dialyzed against buffer E  for use in downstream experiments. Purification was evaluated by SDS-PAGE.Codon-optimized pET-M11 plasmids encoding the N-terminal core (amino acids 93–426) of human NCBE (SLC4A10) isoform B (uniprot ID: 40 in XIA241. Molecular replacement (MR) was performed with phaser in CCP442. A truncated version of the cdb3 crystallographic model (PDB ID: 1HYN)16 was used as the MR search model. The model was built using Coot43 and refined in PHENIX44. The final data collection and refinement statistics are presented in Supplementary Table Purified ntcNDCBE was crystallized via sitting-drop vapour diffusion at 4 °C (40 mg/mL) with 0.1 M formic acid, pH of 4.4, and 9% w/v PEG 8000 and was cryo-protected with 20% ethylene glycol. X-ray diffraction data were collected from a single crystal at the BESSY II 14.1 beamline. The data were processed using XDS2 (Fisher Scientific), MgCl2, CaCl2 and EDTA. The measurements were performed in triplicate on a Jasco FP8500 spectrofluorometer at 25 °C with a stirring speed of 200 rpm and a 30 sec delay between injection and measurement. The cell path length was 10 mm. The excitation wavelength was 295 nm, and emission spectra were recorded from 310 nm to 500 nm. Data were corrected for the dilution factor. The increase in tryptophan fluorescence (F) yield was calculated at the wavelength corresponding to the maximum intensity of the fully Zn2+-saturated curve (336 nm). The increase in tryptophan fluorescence yield due to Zn2+ titration was calculated using equation . The titrants used were ZnClbound is F when the construct is fully saturated, and Ffree is F without any metal added. The decrease in tryptophan fluorescence yield due to EDTA titration was calculated using equation  Hill equation, equation :3\documeKD is the dissociation constant.where B is the maximum value of F, h is the Hill coefficient, and 2+ were determined by ITC in a MicroCal ITC200 system  at 25 °C. The sample cell was filled with 200 µL of degassed protein solution at 50 µM. The titration solution of 0.9–1.5 mM metal in dialysis buffer was titrated in 2 µL injections with a 90-sec delay between injections. Background subtraction was performed using the appropriate buffer. Data integration and curve fitting were performed with the Origin ITC analysis package. ITC experiments were repeated at least twice for each construct, with similar results in both the number of sites and the affinity range.Purified, EDTA-treated ntcNDCBE and ntcNCBE were dialyzed against buffer containing 100 mM NaCl, 20 mM BIS-TRIS pH 7.2, and 0.2 mM TCEP. The affinities of the constructs for Zn45. Based on sequence conservation, ntcNDCBE was divided into VR1 (1–39); CR1 (40–128); VR2 (129–236); and CR2 (237–344). The resulting alignment was mapped onto ntcNDCBE and the transmembrane domain of AE1  using the online server ProtSkin46. The residue conservation score was based on sequence identity.Representative isoforms of each human NBC and AE of the SLC4 family were aligned using MUSCLEQ2Y0W8-4; NBCn1 – Q9Y6M7-1; NCBE – Q6U841-1; NBCe1 – Q9Y6R1-1; NBCe2 – Q9BY07-1; AE1 (1HYN/4KY9) – P02730-1; AE2 – P0492-1; AE3 – P48751-1.Uniprot identifiers: NDCBE – Slc4a10-transfected NIH-3T3 fibroblasts were previously described47. The cells were stably transfected with a plasmid containing the full length mouse slc4a10 construct . Cells were cultured in Dulbecco’s modified Eagle’s medium glutaMAXTM supplemented with 10% donor bovine serum. For pHi measurements cells were grown on glass coverslips.4Cl prepulse for 5 minutes followed by a Na+ free bicarbonate buffered salt solution ). The Na+ dependent HCO3− transport was determined as the dpHi/dt after switching to a bicarbonate buffered Na+ containing solution (BBS) containing 10 µM EIPA to inhibit Na+/H+ exchange activity. Each measurement ended in a one-point calibration at pH 7.0 in a high K+-solution containing 10 µM nigericin. In separate experiments, the fluorescence ratio was calibrated to pH by clamping pHi stepwise from 6–7.5 in a high K+ HBS with 10 µM nigericin48.Prior to measurements the cells were loaded with 2 µM BCECF-AM (ThermoFisher scientific) in a HEPES-buffered salt solution for 10 minutes at 37 °C and mounted in a closed perfusion chamber (Warner Instruments). The chamber was perfused with a linear flow rate of ~0.8 mm/s at 37 °C and placed on an inverted microscope stage in a 37 °C dark box. The fluorophore was excited with alternating 495-nm and 440-nm light from a monochromator (Till photonics). The light emission at 510–535 nm was recorded by a 12-bit cooled monochrome CCD camera  and data was collected from regions of interest of a minimum of 3 cells per coverslips after background correction. Cells were allowed to equilibrate to a baseline level in HEPES buffered salt solution (HBS) followed by acidification by a 20 mM NHHBS: 145 Na+, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 139 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 HPO42−; 20 mM NH4Cl solution: 20 NH4Cl, 125 Na+, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 139 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 PO42−; Na+free BBS: 24 HCO3−, 24 choline, 121 N-Methyl D glucamine, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 115 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 HPO42−; BBS: 24 HCO3− 145 Na+, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 115 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 PO42−. All solutions were adjusted to pH 7.4. Bicarbonate buffers were bubbled with 5% CO2. High K+HBS: 10 Na+, 139 K+, 1.8 Ca2+, 0.8 Mg2+, 139 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 HPO42−.The solutions contained (in mM): i of rat mesenteric small arteries was measured as described previously50. Eleven week old rats were killed by CO2 inhalation where after a segment of a rat mesenteric small artery  was dissected free and mounted in a myograph for isometric force recording. The arteries were kept in a solution (PSS) containing (in mM): Na+ 143, 4 K+, HCO3 25, 1.6 Ca2+, 1.0 Mg2+, Cl 130, 2 HPO42− 2.0, 10.0 HEPES, glucose 5.0). The solution was gassed with 5% CO2 in air and the temperature kept at 37 °C. VSMC pHi was measured using wide-field microcopy of arteries loaded with 5 µM BCECF-AM (Invitrogen) for 30 min. The preparation was placed on the stage of a microscope and the fluorophore excited with alternating 435 nm and 488 nm light. The emission at 510–535 nm at each excitation wavelength was recorded by a photometer and the ratio of the emissions corresponding to excitation at 488 and 435 nm determined. Intracellular acidification was induced by adding 15 mM NH4Cl for 15 min followed by washout into a Na+ free PSS (NaCl was substituted with equimolar amounts of N-methyl-D-glucamine titrated with HCl while NaHCO3 was substituted with equimolar amounts of choline bicarbonate). After 5 min Na2+ was washed in (the Na+ free PSS substituted with PSS) and the rate of increase of the emission ratio was taken as a measure of pH recovery. This value was determined under control conditions, in the presence of 10 µM Zn2+ and again after washout of Zn2+. The pH recovery in the presence of Zn2+ was compared to the mean pH recovery in the control condition and after washout of Zn2+.pHSupplementary data


Candida albicans is an opportunistic fungus responsible for the majority of systemic fungal infections. Multiple factors contribute to C. albicans pathogenicity. C. albicans strains lacking CaArv1 are avirulent. Arv1 has a conserved Arv1 homology domain (AHD) that has a zinc-binding domain containing two cysteine clusters. Here, we explored the role of the CaAHD and zinc-binding motif in CaArv1-dependent virulence. Overall, we found that the CaAHD was necessary but not sufficient for cells to be virulent, whereas the zinc-binding domain was essential, as Caarv1/Caarv1 cells expressing the full-length zinc-binding domain mutants, Caarv1C3S and Caarv1C28S, were avirulent. Phenotypically, we found a direct correlation between the avirulence of Caarv1/Caarv1, AHDCaarrv1, C3SCaarv1, and C28SCaarv1 cells and defects in bud site selection, septa formation and localization, and hyphal formation and elongation. Importantly, all avirulent mutant strains lacked the ability to maintain proper sterol distribution. Overall, our results have established the importance of the AHD and zinc-binding domain in fungal invasion, and have correlated an avirulent phenotype with the inability to maintain proper sterol distribution. Candida albicans and Candida glabrata are pathogenic fungi responsible for the majority of systemic candidiasis cases (is cases . Both aris cases . Our hypSaccharomyces cerevisiae ARV1 (Are1 Are2 required for viability) was identified in a genetic screen looking for recessive alleles toxic to are1 are2 cells  during yeast mating . All expression plasmids contained 500 bp of the endogenous CaARV1 promoter, and 500 bp of the CaARV1 terminator. CaARV1 (CaARV1-5 COMP and CaARV1-3 COMP), CgARV1CaARV1 (CgARV1-5 COMP and CgARV1-3 COMP), and AHDCaARV1  alleles were generated by PCR. To integrate CaARV1 alleles into a Caarv1/Caarv1 homozygous deletion strain, each individual allele was PCR-amplified containing NotI sites. Thus, all Caarv1/Caarv1 strains expressing various arv1 alleles are heterozygous for each allele. PCR fragments were cloned into the disruption plasmid, pDDB78-HIS1 . Full-length CaARV1 was used to construct site-directed mutants . Heterozygous Caarv1/Caarv1 transformants were selected on synthetic minimal medium lacking uracil, arginine, and histidine, thus obtaining +URA3, +ARG4, and +HIS1 transformants. Integration was verified by PCR. All strains were integrated with all selectable markers  to eliminate auxotrophy-specific pleiotrophic effects. All mutant plasmid constructs were sequenced to verify the presence of individual mutations. qRT-PCR indicated that there were no copy differences in the expression of the alleles. Point mutations were generated using the QuickChange Site Directed Mutagenesis Kit (Stratagene) and pDDB78-HIS1-CaARV1 (pHIS1) as a template. The endogenous CaARV1 promoter drove expression of all constructs.Yeast transformations were performed using the Frozen EZ Yeast Transformation II Kit (Zymo Research). All ng BWP17 . The PCRCaarv1/Caarv1 cells expressing CgARV1, the full-length coding sequence of CgARV1 was subcloned into pDDB78-HIS1 containing 500 bp of the CaARV1 promoter, full length CgARV1 (1000 bp), and 500 bp of the CaARV1 terminator. Caarv1/Caarv1 cells expressing AHD were generated by integrating pDDB78-HIS1 containing the CaARV1 AHD domain.To construct Protein extraction was performed as described previously . ProteinCandida albicans strains were grown for 18 hr in YEPD at 37°, 200 rpm; ∼15 ml of culture was harvested and cells were washed twice with ddH2O. Cells were then resuspended in 1 ml ddH2O and split equally into two tubes, one sample for the determination of the dry weight of cells, the other for sterol extraction.Single colonies of Nonsaponifiable lipids were prepared and extracted as reported previously  at 30°, harvested by centrifugation, washed twice with 1× phosphate-buffered saline (PBS), counted by hemocytometry, and resuspended in 1× PBS at the required density. For survival experiments, mice were injected via the tail vein with 200 µl of 1 × 104 cells/ml of C. albicans in 1× PBS. Infected animals were monitored daily for 30 d postinfection, and were considered moribund when they could no longer reach food or water. Moribund animals and mice surviving to the end of the study were killed by CO2 asphyxiation, and survival times were recorded. All animals were housed at Temple University—an Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) accredited facility. The Temple University Institutional Animal Care and Use Committee (IACUC) approved the protocol.Female BALB/cJ mice (Jackson Labs) aged 6–8 wk, weighing ∼18–22 g, were housed in groups of as many as four animals, and were supplied food and water 5 cells/ml of C. albicans in 1× PBS. Animals were killed 48 hr postinfection. Concentrations of yeast inocula were determined by plate viability counts made from organ suspensions. A total of 10 mice/strain was infected for survival and organ fungal load experiments. Experimental procedures were carried out according to the National Institutes of Health (NIH) guidelines for the ethical treatment of animals. Temple University’s IACUC approved all animal use protocols.For organ fungal load determination, mice were injected via the tail vein with 200 µl of 5 × 10C. albicans were killed 48 hr postinfection, and target organs  were removed aseptically and homogenized in 4 ml of 1× PBS. Fungal load was determined by making 10-fold serial dilutions in 1× PBS, and plating 40 μl on YEPD plates containing 34 μg/ml chloramphenicol. Plates were incubated at 30° for 24 hr. Total CFUs were determined, and counts were expressed as the log10 CFU/organ weight in grams; 8–10 livers were combined and analyzed.Mice infected with Hyphal formation was induced at 37° for 3 hr in 10% fetal bovine serum (FBS). Cells were fixed with 2% paraformaldehyde for 10 min at room temperature, followed by gentle washing with PBS. Cells were stored at 4° until microscopic analysis. Bud scars were visualized by calcofluor white staining , with an incubation of 2–5 min at room temperature. Microscopy was performed immediately with 100× magnifications using a Leica fluorescence microscope with an attached camera. At least 300 cells were examined, and the data are the average of five independent experiments.600 U/ml. After 10 min of mixing at 23°, fixed cells were centrifuged, and the pellet was washed twice with 10 ml distilled water. Washed cells were resuspended in 1 ml of water; 200 µl was mixed with 4 µl of freshly made 5 mg/ml filipin complex in ethanol . After incubating in the dark for 15 min, cells were spotted directly onto slides, and filipin fluorescence was observed with a UV filter set using neutral density filters. For all fluorescence microscopy experiments, samples were mounted on microscope slides, sealed under coverslips with nail polish, and imaged on a Leica fluorescence microscope with an attached camera. Three hundred cells were counted for each strain, and the data are the average of five independent experiments.Unesterified sterol was visualized using filipin staining. One milliliter of 37.5% formaldehyde was added to 9 ml of cell culture grown to a density of 0.7 ODStrains and all reagents are available upon request.Scarv1 cells .Immunoblot analysis showed that the h CaArv1 . qRT-PCRCaARV1/CaARV1 cells were dead by d 18, with 50% lost by d 6 (Caarv1/CaARV1 cells were dead by d 5 (P < 0.0001) (Caarv1/Caarv1 cells survived until the study was terminated at 30 d (P < 0.0001). These results are in good agreement with previous work .Mice injected with  0.0001) . Twenty C3SCaarv1) and Cys28 (C28SCaarv1) were mutated in the first and second cysteine clusters of the zinc-binding domain . Mice injected with either C3SCaarv1 .Next, we tested the role of the AHD zinc-binding domain in virulence. In this case, amino acids were changed in full-length CaArv1. Cysteines at positions Cys3 . The remaining mice were dead by d 10.Finally, we explored the conservation of CaARV1 and CgARV1 alleles.Our results together indicated that the AHD alone does not possess the same function as full-length CaArv1. They also show that zinc-binding domain function is needed to confer virulence, while indicating a degree of conservation between Multiple tissue failure contributes to the mortality associated with disseminated candidiasis, as organ colonization and invasion is normally seen during an invasive infection . Thus, fCaARV1/CaARV1- and Caarv1/CaARV1-injected mice . These results are in good agreement with previous results . Heterozygous cells had a higher percentage of ergosterol (130%), and decreased percentages of zymosterol (28%), episterol (25%), fecosterol (23%), ergosta-5,7,24(28)-trienol (23%), and lanosterol (10%) compared to CaARV1/CaARV1 cells , and a lower percentage of zymosterol (26%) compared to CaARV1/CaARV1 cells.Interestingly, the sterol compositions of V1 cells . Caarv1/AHDCaarv1, C3SCaarv1, or C28SCaarv1 alleles. Sterol intermediates levels were compared to Caarv1/CaARV1 cells. AHDCarv1 cells had a higher percentage of ergosta 5,7 dienol (246%) and a lower percentage of 4,4-dimethylzymosterol (10%), whereas both C3SCarv1 and C28SCarv1 cells had higher percentages of fecosterol (415%), ergosta-5,7,24(28)-trienol (630%), ergosta 5,7 dienol (454%), and lanosterol (242%). The sterol composition of CgARV1Carv1 cells was similar to that of Caarv1/CaARV1 cells.To next examine if the AHD, Cys3, and Cys28 were required for maintaining normal sterol composition, sterol content was determined in cells expressing Caarv1/Carv1 (300%), AHDCaarv1 (246%), C3SCarv1 (630%), and C28SCarv1 (630%)].In looking at the data as a whole, it is interesting that all avirulent strains accumulated the same sterol intermediate, ergosta 5,7 dienol: [Hyphal formation and subsequent elongation are necessary for strains to be virulent . Cells wCaarv1/CaARV1 cells did have a reduction in the number of cells forming hyphae compared to CaARV1/CaARV1 cells (P < 0.001). The percentages of Caarv1/Caarv1, AHDCaarv1, C3SCaarv1, and C28SCaarv1 cells forming hyphae were also significantly reduced . Caarv1/Caarv1 cells expressing CgARV1 did not display a reduction in hyphal formation.Interestingly, in vitro after cells were grown in hyphae-inducing medium for 3 hr.Bud site selection along the mother cell periphery dictates where hyphal formation will initiate . These mutants had constrictions along the germ tube, which lacked chitin staining (Caarv1/Caarv1 cells was that they had a second chitin bud site that was the initiating point for another germ tube (AHDCaarv1 (∼95%), C3SCaarv1 (90%), and C28SCaarv1 (85%) cells had only a single chitin bud site that remained at the initial mother–daughter neck . Caarv1/Caarv1, AHDCaarv1, C3SCaarv1/Caarv1, and C28SCaarv1/Caarv1 cells all showed defects in sterol distribution . The percentage of AHDCaarv1 cells with hyphal tip-localized sterol was lower than that seen for Caarv1/Caarv1 cells (20%) . Caarv1/C3SCaarv1 and C28SCaarv1 cells had the least number of cells localizing their sterol to the hyphal tip .ribution , localizribution , arrows.ribution . The perV1 cells . Caarv1/ls (20%) . Caarv1Aphal tip , ∼10%. Tphal tip . FinallyCaarv1/Caarv1, AHDCaarv1, C3SCaarv1/Caarv1, and C28SCaarv1/Caarv1 cells lack virulence. These strains also had sterol distribution defects and accumulated what appeared to be sterol aggregates. Thus, we can conclude that there was a direct correlation between cells being avirulent and their lack of ability to properly distribute their sterol during hyphal growth.Thus, C. albicans strains lacking CaArv1 are avirulent, suggesting that Arv1 function has a role in maintaining virulence. CaArv1 contains a CaAHD domain that has within it a zinc-binding motif. Here, we explored whether the CaAHD alone was responsible for the virulence function of CaArv1, and, if so, was the zinc-binding motif necessary for virulence. The CaAHD alone could not replace full-length Arv1 function, suggesting that additional domains outside the AHD play a role in virulence. However, we did find that the CaAHD zinc-binding motif was needed for virulence, as cells containing an intact CaArv1 protein harboring either a Cys3 or Cys28 mutation were avirulent, substantiating the hypothesis that CaAHD function is necessary for virulence but is not sufficient. These data hint at the possibility that the activity of the zinc-binding motif is the critical function associated with CaAHD.AHDCaarv1, C3SCaarv1, and C28SCaarv1 cells all had defects in sterol distribution and septa formation, and all lacked the ability to localize their sterol to the growing hyphal tip. Interestingly, these mutant strains accumulated several sterol biosynthetic intermediates when compared to wild-type cells. S. cerevisae cells lacking ScArv1 have elevated sterol and phosphatidylserine levels in their plasma membrane . Whether this increased secretion contributes to increased pathogenicity is presently being explored.Organ colonization and invasion are considered major mortality factors, especially in the case of the kidney . The orf 19.1212 protein product has a FFAT domain (S. cerevisiae and virulence in C. albicans (Data suggest that Arv1 has the ability to distribute lipids other than sterol, including the glycerophospholipid, phosphatidylserine. C. albicans Arv1 are required for function and virulence. They also indicate that the CaAHD is necessary for virulence, but it alone cannot substitute for full-length CaArv1. Moreover, we have validated the importance of the zinc-binding domain in conferring virulence. AHD homology searches indicate that the AHD and zinc-binding domain are conserved among a large population of pathogenic yeasts. Thus, targeting Arv1 for drug discovery may represent a novel approach for treating systemic candidiasis.Overall, our data strongly suggest that multiple domains of


Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B Aspergillusflavus and Aspergilus parasiticus [1 (AFB1) is the most toxic and widely distributed of all identified aflatoxins [1 is not toxic in its native form, but highly activated when converted to electrophilic AFB1 exo-8,9-epoxide (AFBO) in vivo [1 [1 in human [1 and to activate the carcinogen at relative lower substrate concentrations [1 activation based on the following experimental facts: (i) CYP3A4 is far more abundant than CYP1A2 in the human liver [1 into exo-8,9-epoxide and a detoxification product AFQ1 [1 into detoxification product AFM1, exo- and endo-8,9-epoxides [1 activation. For instance, CYP3A37 in the turkey liver [1 into AFBO. Besides CYP1A2 and CYP3A4, it has been reported that human CYP2A6, CYP2A13, and CYP2B7 are also active in converting AFB1 to active metabolites [1 activation in vivo. CYP3A5 is another important human CYP3A isoform, which shares 85% amino acid sequence identity with CYP3A4 [1 into AFBO [Aflatoxins are secondary metabolites mainly produced by asiticus . They arasiticus . Aflatoxlatoxins ,4. AFB1  in vivo ,6. AFBO  vivo [1 . Extensiin human , though in human ,11,12,13an liver ; (ii) CYuct AFQ1 ; and . Using LC-MS/MS  and the lowest binding energy  and a lower binding energy  and three quails were used from Animal Husbandry Institute, Guangdong Academy of Agricultural Sciences. The quail livers were isolated to make the microsomes. The animals were sacrificed by injection of excess sodium pentobarbital, and then the surgeries of liver separation were performed.1 was purchased from Fermentek . E.Z.N.A® Total RNA Kit was purchased from Omega Bio-Tek . Prime Script RT reagent kit and restriction endonucleases were obtained from Takara BIO Inc. . High fidelity DNA polymerase KOD-Plus- and KOD FX were from Toyobo . QuikChange site-directed mutagenesis kit was purchased from Stratagene . Isopropyl-β-d-thiogalactoside (IPTG) and NADPH were from Gen-View Scientific Inc. . Nifedipine (>98% purity), oxidized nifedipine (~95% purity) standards, NADPH cytochrome P450 oxidoreductase (94% purity), and cytochrome b5 (≥90% purity) were purchased from Sigma-Aldrich Corporation . A 5-mL HisTrap HP column was purchased from GE Healthcare . A ZORBAX SB-C18 column  was purchased from Agilent Technologies . Chromatographic grade acetonitrile, methanol, and formic acid were obtained from Merck . All the primers were synthesized in Invitrogen Biotechnology . All the other reagents and chemicals used were of analytical grade available.AFBCYP3A29 (NCBI NM_214423), the forward primer 5′-GGAAAATCCGAGGAGAGAATCA-3′ and the reverse primer 5′-CATCAAAGCCCAAGTCCTTAGAG-3′ were designed to amplify the interest gene. PCR reaction was performed using a thermocycling program that ran for 2 min at 94 °C and then proceeded for 32 cycles at 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1.5 min. The PCR product was ligated into the pMD 19-T vector through TA cloning. The CYP3A29 gene sequence was verified by DNA sequencing. Subsequently, CYP3A29 gene was subcloned into the pCW Ori+ expression vector. To allow functional expression in E. coli, the cDNA fragment encoding the bacterial ompA leader sequence were fused to CYP3A29 by PCR in frame with the initiation codon [CYP3A29 gene. The resulting plasmid was confirmed by DNA sequencing and named pCW/3A29.Total RNA was isolated from porcine liver using a Total RNA kit. The first-strand cDNA was synthesized from 2 μg of total RNA using a Prime Script RT reagent kit. According to the published mRNA sequence of pig on codon . The leaThe pCW/3A29 expression vector was used as the plasmid DNA template for oligonucleotide-directed mutagenesis. According to our preliminary docking results and other related reports ,55, the E. coli DH5α cells. A single colony containing the recombinant plasmid was inoculated and cultured in 500 mL of TB broth containing 100 μg/mL ampicillin, 1 mM thiamine, and a mixture of trace elements. The cells were grown at 37 °C until the absorbance at 600 nm reached 0.8. Then, the expression was induced at 30 °C by the addition of 1 mM isopropyl β-d-thiogalactoside (IPTG). After 36 h, the cells were harvested and treated by 0.25 mg/mL of lysozyme. The techniques used for cell disruption, preparation of spheroplasts, and isolation of membranes were as described previously [Recombinant CYP3A29 and the mutants were expressed in 2+∙CO vs. Fe2+ difference spectra of the recombinant CYP3A29 and the mutants were collected as described previously [450–490 = 91 mM−1∙cm−1 [Feeviously  on a UV-M−1∙cm−1 .Far-UV CD spectra of the recombinant CYP3A29 and the mutants were collected on a Chirascan spectrometer . The measurement parameters and methods employed were described as previously . The con5, 0.02 mg/mL liposomes , 0.2 mg/mL sodium cholate, 3 mM glutathione, 30 mM MgCl2, and 200 μM nifedipine in 100 mM potassium phosphate buffer (pH 7.4), making a final volume of 200 μL. The reaction was initiated by the addition of 1 mM NADPH  and performed at 37 °C for 10 min, before being terminated by 2 mL of cold methylene chloride. After being added into 100 μL of 2 M sodium carbonate buffer (pH 10.5), the mixtures were centrifuged at 3000 g for 10 min. The combined organic layers were transferred and evaporated to dryness under a stream of high-pure nitrogen. The residue was redissolved in 150 μL of methanol and analyzed by HPLC. HPLC analyses were performed on a Waters Alliance system equipped with 2695 separation module. A symmetry reverse-phase C18 column was used to analyze the substrates nifedipine and their metabolites. The injection volume was 20 µL. An isocratic elution with 55% (v/v) methanol in water was applied to separate the substrates and the metabolites with a flow rate of 1 mL/min at 30 °C. Elution was monitored by the UV absorbance at 254 nm. The nifedipine and oxidized nifedipine standards were used as the controls to identify the substrate and the product.Reconstitution of a nifedipine-oxidized system followed the method of Shaw et al.  with som5, 0.02 mg/mL liposomes , 0.2 mg/mL sodium cholate, 3 mM glutathione, 30 mM MgCl2, 50 mM Tris, and 8 μM AFB1 in 100 mM potassium phosphate buffer (pH 7.4). Subsequently, the mutants S119A, F304A and T309A were incubated in the reconstitution system without ketoconazole. The reaction was initiated by the addition of 1 mM NADPH  and performed at 37 °C for 3 h prior to termination by addition of 200 μL ice-cold methanol spiked with 0.76 μM aflatoxin G1 as an internal standard. The samples were kept at −20 °C overnight to facilitate protein precipitation and then centrifuged at 10,000 g for 15 min prior to analysis [1-8,9-dihydrodiol generated in this incubation system reacted with Tris to form a highly fluorescent compound [The recombinant CYP3A29 was incubated in the presence or absence of 5 μM ketoconazole with 0.1 μM NADPH cytochrome P450 oxidoreductase, 0.1 μM cytochrome banalysis ,64. The compound . Quail lcompound . The maj1 metabolites produced by CYP3A29 and the mutants were detected on a Waters Alliance e2695 liquid chromatography system  equipped with a 2475 fluorescence detector. Samples were separated at 25 °C on a ZORBAX SB-C18 column. The mobile phase, the gradient schedule, and detection condition were described as previously [The AFBeviously . The flo2-terminal region of CYP3A29 (residues 3 to 24) was deleted. The quality of the resultant model structure was analyzed and evaluated by PROCHECK, What_check, Verify_3D, Errat, and Prove software .Because of the high sequence identity of CYP3A29 with human CYP3A4 (77%), the three-dimensional structure model of CYP3A29 was built using SWISS-MODEL server as a homology modeling tool ,66,67, b1 to CYP3A29. The AutoDock 4.2 docking program was used to find the best configurations for receptor and ligand complex [1 ligand was flexible, in which only one bond was rotatable. To cover the whole active site of CYP3A29, the grid box was set to 60 × 60 × 60 points with spacing of 0.375 Å. The box center was set at 60.055, 76.691, and 9.028. AutoDockTools 1.5.6  was used to add Gasteiger charges to the CYP3A29 coordinates and AFB1. The Lamarckian genetic algorithm was used to determine the globally optimized conformations and orientations of the ligand with minimized free energy. All the docking simulations were performed with 100 cycles of running using an initial population size of 150 and a maximum of 2.5 million energy evaluations. The other parameters were set at their default values. Docking results were clustered using a root mean square deviation (RMSD) of 2.0 Å and were ranked according to the change in free energy (ΔG) estimated by AutoDock. Conformations with the lowest free energies of binding were selected as the best binding modes. Molecular graphics were generated by PyMOL [A molecular docking study was conducted to construct a binding model of AFB complex . For eacby PyMOL .t test and p < 0.05 was considered significant.Resulting data are reported as the mean and standard error of means (Mean ± SE) based on at least three independent experiments. Statistical significance of differences between means was assessed by two-tailed


The cytostatic potentials of the PSG toxin were determined by the lymphocyte migration inhibition assay. The PSG toxin (50 μg/ml) exhibited commendable inhibition of the migration of lymphocytes across the agarose gel matrix under the presence of lipopolysaccharide mitogen, with a mean migration index of 0.625. The cytotoxicity of the PSG toxin against selected cancer cell lines was determined using the MTT assay. The PSG toxin exhibited dose-dependent cytotoxicity against the MCF-7 breast cancer cells followed by KB , HeLa  and A549 (lung) cancer cell lines. The PSG toxin also exhibited proportional release of LDH leakage by mitochondrial damage with an IC50 of 13.85 μM against MCF-7 breast cancer cells. The in vitro anticancer activity of the PSG toxin against the selected cell lines was evaluated by Karthik et al. (2017) [1].The data presented illustrated the  Specifications TableValue of the data•S. pharaonis against peripheral blood mononuclear leucocytes.The provided data demonstrates the cytostatic potentials of the PSG toxin from •S. pharaonis against the selected cancer cell lines.The data provided illustrates the commendable anti-proliferative action of the PSG toxin from •The data might be valuable to researchers interested in anti-proliferative action of toxins from marine mollusks.•The data might also be of value to researchers investigating the anti-proliferative and anticancer activity of marine bioactive compounds.1The migration of the lymphocytes was inhibited by the PSG toxin  with a mean diameter of 1.65±0.55 mm and 1.42±0.64 mm respectively. The lymphocytes exhibited pronounced migration under the influence of LPS with a mean diameter of 3.45±0.83 mm .Fig. 1Cy50 concentration of 32.5 µM. The purified PSG toxin also exhibited significant inhibition against the proliferation of HeLa cervical cancer cells and A549 lung cancer cells at an IC50 value of 16.5 µg/ml (23.2 µM) and 22.45 µg/ml (31.65 µM)  . The inl50 concentrations of 22.8 μg/ml, 17.45 μg/ml and 23.52 μg/ml respectively  was determined using the lymphocyte migration inhibition assay following the method of Mousseau et al. (2007). The inhibition of leucocyte migration treated with PSG toxin  under the influence of mitogen (lipopolysaccharide) was measured using a microscope ruler 2.1.250) were determined.The cytotoxicity of the purified PSG toxin against selected cancer cell lines was determined by the MTT cell viability assay 2.1.3The cell viability and membrane permeability of the PSG toxin  against the KB, HeLa and A549 cancer cells was determined using the LDH leakage assay


Proteobacteria is the genetically encoded contact-dependent type VI secretion system (T6SS), a weapon used to kill heteroclonal neighbors by direct injection of toxic effectors. Here, we experimentally demonstrate that Vibrio cholerae can acquire new T6SS effector genes via horizontal transfer and utilize them to kill neighboring cells. Replacement of one or more parental alleles with novel effectors allows the recombinant strain to dramatically outcompete its parent. Using spatially explicit modeling, we examine how this process could affect the ecology and evolution of surface-attached microbial populations. HGT of T6SS effector-immunity pairs is risky: transformation brings a cell into conflict with its former clone mates but can be adaptive when superior T6SS alleles are acquired. More generally, we find that these costs and benefits are not symmetric and that high rates of HGT can act as a hedge against competitors with unpredictable T6SS efficacy. We conclude that antagonism and horizontal transfer drive successive rounds of weapon optimization and selective sweeps, dynamically shaping the composition of microbial communities.Horizontal gene transfer (HGT) can have profound effects on bacterial evolution by allowing individuals to rapidly acquire adaptive traits that shape their strategies for competition. One strategy for intermicrobial antagonism often used by  Proteobacteria to kill adjacent competitors. While DNA released by T6 killing can be horizontally acquired, it remains untested whether T6 genes themselves can be horizontally acquired and then utilized to compete with neighboring cells. Using naturally transformable Vibrio cholerae, we provide the first direct empirical support for the hypothesis that T6 genes are exchanged horizontally  and functionally deployed to compete with neighboring cells. Using computational simulations, we also demonstrate that high rates of HGT can be adaptive, allowing V. cholerae to improve upon existing T6 weaponry and survive direct encounters with otherwise superior competitors. We anticipate that our evolutionary results are of broad microbiological relevance, applying to many bacteria capable of HGT that utilize the T6SS or similar antagonistic systems, and highlight the profound impact of HGT in shaping microbial community structure.The contact-dependent type VI secretion system (T6SS) is frequently used by  Vibrio cholerae, which often carries the cholera toxin (CTX)-encoding prophage responsible for major cholera outbreaks in Haiti and regions of endemicity (–V. cholerae isolates from aquatic environments are invariably nontoxigenic (CTX−) and commonly associated with chitinous surfaces like shells of crabs or zooplankton that may promote transmission  by plasmid conjugation, viral transduction, and natural transformation plays a fundamental role in the evolution of bacteria and archaea and also in plants and other eukaryotes . Genomicdemicity –5. V. chsmission .V. cholerae encode a type VI secretion system (T6SS) weapon that can deliver toxic effector proteins directly into grazing predators, diverse proteobacteria, and other V. cholerae bacteria that lack cognate immunity proteins  or ΔcomEA (Com−) nontransformable derivatives were used as recipients in standard transformation assays in artificial seawater with induction by chitin, using 692-79 donor DNA marked with antibiotic resistance at the Aux1  or Aux2  locus. Inheritance of the donor T6SS Aux1 or Aux2 operon in resistant transformants was verified by colony PCR. (B) Recipients were cocultured with donor strains derived from 692-79 that were marked with antibiotic resistance at the Aux1  or Aux2  locus in artificial seawater supplemented with chitin. Donor strains were also inactivated for T6SS . Bars denote means ± standard deviations . Download FIG S1, TIF file, 0.1 MB.T6SS operons can be acquired by natural transformation. (A) Copyright © 2017 Thomas et al.2017Thomas et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the F3,12 = 241.5, P < 0.0001 [+ experiments in F3,12 = 983.2, P < 0.0001 [vgrG-3 in the double  transformant completely abolished killing of the donor 692-79 , the Aux2 operon (A2), and both operons  are able to kill C6706 to an increasing degree in standard 3-h T6 killing assays . We prer 692-79 . Replace< 0.0001 ) but mad< 0.0001 ).10.1128/mBio.00654-17.2FIG S2 − ΔvasK “prey” strains were mixed at a 10:1 ratio and incubated at 37°C for 3 h on membrane filters, and surviving prey CFU were counted on selective medium. Killer strains were either T6SS+ (gray bars) or T6SS−  and had a functional VgrG-3 main cluster effector (A) or were deleted for vgrG-3 (B) Strains used were C6706 (recipient), 692-79 (donor), and C6706 derivatives in which either Aux1 (A1), Aux2 (A2), or both clusters  were replaced by those of 692-79. Bars denote means ± standard deviations . Download FIG S2, TIF file, 0.1 MB.VgrG-3 is responsible for residual T6SS-killing activity of the T6SS Aux1 and -2 replacement strain toward its donor. “Killer” strains and T6SSCopyright © 2017 Thomas et al.2017Thomas et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Vibrio cholerae V52 (and C6706) were the most potent in pairwise competition experiments among the different allele combinations tested , Aux2 (A2), or both Aux1 and 2  replacement had increased fitness over both recipient and donor  or T6SS− (B). Symbols denote mean ± standard deviation . Download FIG S3, TIF file, 0.1 MB.Horizontally acquired T6SS alleles are beneficial in long-term competition assays. Derivatives of C6706 were mixed in a 1:1:1:1 initial ratio, allowed to compete on nonselective solid agar for 24 h at 37°C, and resuspended, and surviving CFU were counted on selective medium. The suspension was repeat passaged on solid agar for 5 days. Strains used were C6706 (Rec), 692-79 (donor), and C6706 derivatives in which either Aux1 (A1), Aux2 (A2), or both clusters  were replaced by those of 692-79. All strains were either T6SSCopyright © 2017 Thomas et al.2017Thomas et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Our experimental results highlight a specific scenario of transfer of T6SS clusters. In order to derive general principles for how natural selection might act on the HGT of T6SS clusters, we extended a model that we previously developed to consider growth and competition on a two-dimensional lattice between any two mutually antagonistic strains . In our 10.1128/mBio.00654-17.5MOVIE S1MOVIE S1, AVI file, 7.8 MB.Visualization of the dynamics of HGT and T6SS. This simulation examines HGT under favorable conditions: the population is initially highly structured and the competitor possesses more efficacious T6SS alleles (3-fold better than the recipient). Recipient cells that take up one allele from the blue competitor are shown in green, those that have taken up two alleles are shown in orange, and those that have swapped out all three of their T6SS alleles are shown in yellow. Transformants possess a strong within-patch selective advantage, leading to repeated waves of HGT and T6SS-mediated sweeps until all of the recipient cells possess their competitors’ T6SS effector alleles (yellow). In this simulation, each cell had a 0.0005 probability of undergoing HGT per time step. Download Copyright © 2017 Thomas et al.2017Thomas et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the y = −74.2x + 0.991, r2 = 0.99), was very costly, reducing fitness by 40% when the per-cell probability of HGT per time point was 0.005.T6SS-mediated conflict is inherently risky, as competitors vary widely in their relative killing efficacy . While HThe fact that HGT incurs significant costs due to intrastrain conflict and benefits via survival when facing a superior competitor raises the possibility that it may act as a hedge against uncertainty in the T6SS efficacy of future opponents. We examined this hypothesis by calculating a recipient strain’s geometric mean fitness across a pair of interactions in which it faced a competitor with symmetrically varying T6SS efficacy  and across a range (0 to 0.005) of HGT. A geometric mean fitness above 1 indicates that HGT is adaptive. When the two strains have T6SSs with similar efficacies, the costs of HGT (intrastrain conflict) exceed its benefits and selection acts against HGT . However+ strain is facing a competitor with far superior T6SS alleles. Indeed, we show that variability in opponent T6SS allele quality alone can select for high rates of HGT, allowing HGT to act as a hedge against uncertain opponent quality.We have demonstrated that toxigenic bacteria can acquire T6SS effector-immunity pairs from nontoxigenic neighbors via natural transformation, consistent with the hypothesis that this frequently occurs in the environment , 18. OncV. cholerae that appear to have undergone multiple rounds of additive HGT at a single highly recombinogenic T6SS locus to form “immunity gene arrays” encoding several putative T6SS immunity proteins, which may provide protection against effectors from multiple V. cholerae and non-V. cholerae strains  avoids costs of intrastrain genetic conflict. Thus, additive recombination represents an additional mechanism to promote horizontal spread of T6SS alleles while mitigating the costs of HGT.In our experimental system, we were only able to observe examples of T6SS cluster replacement. However, bioinformatic and phylogenetic approaches have recently identified lineages of  strains . SuccessV. cholerae strains C6706 and A1552, both natural transformation and T6S are coinduced under conditions of chitin utilization, starvation, and quorum sensing at high cell density , spectinomycin (100 µg/ml), streptomycin (5 mg/ml), and diaminopimelic acid (50 µg/ml) where appropriate.All 06 str-2  or envir10.1128/mBio.00654-17.4TABLE S1 V. cholerae strain and plasmid. Download TABLE S1, DOCX file, 0.01 MB.List of strains and plasmids used in this study. The genotype or description of each relevant Copyright © 2017 Thomas et al.2017Thomas et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the V. cholerae were constructed by previously described allelic exchange methods (In-frame deletions and promoter-replacement mutants in  methods . Standar600) of 0.4 to 0.6. Cultures were washed in artificial seawater (ASW) and diluted to an OD600 of 0.15, and 2-ml cultures in ASW were incubated at 30°C with a chitin tile for 24 h. The chitin tile with adherent bacteria was moved to 2 ml fresh ASW containing 1 µg/ml of genomic DNA marked with an antibiotic resistance cassette and incubated at 30°C for a further 24 h. Cells were collected by washing the chitin tile in 2 ml of fresh ASW, and dilutions were plated on selective medium to determine transformation frequency. Acquisition of T6SS alleles from donor DNA linked to the antibiotic resistance cassette was verified by allele-specific multiplex PCR using primers specific for C6706 or 697-79. Transformation in coculture was performed as described above with the following exceptions: both donor and recipient strain were each grown to an OD600 of 0.4 to 0.6 in LB and diluted to an OD600 of 0.15 after washing in ASW mixed in a 1:1 ratio, and 2 ml of each mixed culture was inoculated with a chitin tile at 30°C. No exogenous genomic DNA was added.Assays for natural transformation were performed as previously described . Briefly600 of 1.00. Killer and prey strains were mixed at a ratio of 10:1, and 50 µl of each suspension was spotted onto a gridded filter disc (Whatman) placed on an LB agar plate and confined to 9 grid squares (3.1 by 3.1 mm each) to reduce variability. Filters were incubated at 37°C for 3 h and washed with 5 ml LB to dislodge cells, and dilutions were plated on selective medium to determine prey survival.T6SS killing assays were performed as previously described . Killer 600 of 1.00. Pairs of strains were mixed 1:1, and 50 µl of each suspension was inoculated on LB agar plugs in a 12-well plate, dried under laminar airflow in a biosafety cabinet, and incubated overnight at 37°C. Cells were resuspended in 1 ml LB, and dilutions were plated on selective medium to determine survival of each competitor. Competitive index was determined as final ratio of survivors/initial ratio of inoculation.Competitor strains were grown overnight in LB at 37°C, centrifuged, and resuspended in fresh medium to an ODpost hoc test, with an α of 0.05. Within each experiment , different letters denote significantly different means. The consequence of horizontal acquisition of T6SS effectors for pairwise interactions  with a Bonferroni correction to control for multiple comparisons. Competitive indices were log10 transformed prior to analysis to give equal weight to competitive ratios both above and below 1.All statistics were calculated in JMP 12.0. T6SS efficacy  to D wasractions  was examWe extended the individual-based model from the work of McNally et al. . Square t, divided by their starting ratio. The fitness landscapes shown in these figures were calculated across a 20-by-20 factorial combination of HGT rates and competitor T6SS efficacies for 15,000 time points.Recipient genotype cells had a probability, ranging from 0 to 0.005, of replacing one of their T6SS effector protein-immunity pair alleles with one obtained from the environment at each time step. This was performed in a weighted manner, according to the frequency of recipient versus competitor alleles in the population, and did not depend on the spatial distribution of those cells in the population. Biologically, this is analogous to cells sampling DNA from a well-mixed bulk medium above a surface-attached population. Note that this means that HGT can occur in both directions, with the recipient genotype replacing its T6SS with those from the competitor and, in future HGT events, reverting to its original T6SS genotype. HGT events occur in a stepwise manner, and so only one allele can be replaced per time point. The probability that adjacent cells succeed in killing was calculated as the sum of the per-allele killing probability for all of the T6SS loci that differ between the cells. Simulations were run in Python (code available upon request). Edges (outermost rows/columns) were trimmed prior to analysis. For 10.1128/mBio.00654-17.6MOVIE S2MOVIE S2, MOV file, 9.9 MB.Time-lapse video of the fitness landscape shown in Copyright © 2017 Thomas et al.2017Thomas et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00654-17.7MOVIE S3 MOVIE S3, MOV file, 8.4 MB.Time-lapse video of the fitness landscape shown in Copyright © 2017 Thomas et al.2017Thomas et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We examined whether or not HGT could act as a hedge against unpredictable competitors by simulating pairs of interactions, in which the T6SS killing efficacy of the recipient strain varied symmetrically  around the competitor’s killing efficacy. We calculated the geometric mean of fitness  for these pairs of interactions across a range of HGT rates (0 to 0.005), with five replicates of the simulation run per treatment combination.


The crystal structure of dirubidium hydrogen citrate has been solved and refined using laboratory X-ray powder diffraction data, and optimized using density functional techniques. +·HC6H5O72−, has been solved and refined using laboratory X-ray powder diffraction data, and optimized using density functional techniques. The un-ionized carboxylic acid group forms helical chains of very strong hydrogen bonds (O⋯O ∼ 2.42 Å) along the b axis. The hydroxy group participates in a chain of intra- and intermolecular hydrogen bonds along the c axis. These hydrogen bonds result in corrugated hydrogen-bonded layers in the bc plane. The Rb+ cations are six-coordinate, and share edges and corners to form layers in the ab plane. The interlayer contacts are composed of the hydrophobic methylene groups.The crystal structure of dirubidium hydrogen citrate, 2Rb NaKHC6H5O7, NaK2C6H5O7, Na3C6H5O7, NaH2C6H5O7, Na2HC6H5O7, and K3C6H5O7, have been published recently 2, have been communicated  citrate salts to understand the conformational flexibility, ionization, coordination tendencies, and hydrogen bonding of the anion, we have determined several new crystal structures. Most of the new structures were solved using powder diffraction data (laboratory and/or synchrotron), but single crystals were used where available. The general trends and conclusions about the 16 new compounds and 12 previously characterized structures are being reported separately , and the other is intermolecular. These hydrogen bonds contribute 9.3 and 8.6 kcal mol−1 to the crystal energy.The Rb cation is six-coordinate . The coordination polyhedra share corners and edges to form layers in the ne Fig. 3. The un-ne Fig. 3. The Mulne Fig. 3, this hyet al., 2016Details of the comprehensive literature search for citrate structures are presented in Rammohan & Kaduk 2017. A reduc3C6H5O7(H2O)  was dissolved in 10 ml deionized water. Rb2CO3  was added to the citric acid solution slowly with stirring. The resulting clear colorless solution was evaporated to dryness in an oven at 333 K.HITO/CRYSFIRE , and we believe that P21/m is the correct space group.The structure was solved and initially refined in the space group P21 is consistent with ordered asymmetric hydrogen bonds, while P21/m is consistent with both disordered asymmetric hydrogen bonds or symmetric hydrogen bonds. Crystallographically, it would be difficult to distinguish these two possibilities, especially using X-ray powder diffraction data. DFT calculations on the asymmetric (P21) and symmetric (P21/m) hydrogen-bond models indicate that the symmetric model is 0.2 kcal mol−1 lower in energy. This difference is within the expected error range of such calculations. Since the crystallography strongly indicates the higher symmetry, we believe that the P21/m model with symmetric hydrogen bonds is the best model for this structure.Stoichiometry requires one carboxylic acid proton per citrate. The space group CRYSTAL14  was carried out using  al. 2012, and the al. 2008. The cal10.1107/S2056989016020168/vn2120sup1.cifCrystal structure: contains datablock(s) RAMM020C_publ, ramm020c_DFT. DOI: 1523504, 1523503CCDC references: crystallographic information; 3D view; checkCIF reportAdditional supporting information: