In vivo single-cell high-dimensional mass cytometry analysis to track the interactions between Klebsiella pneumoniae and myeloid cells.

In vivo single-cell approaches have transformed our understanding of the immune populations in tissues. Mass cytometry (CyTOF), that combines the resolution of mass spectrometry with the ability to conduct multiplexed measurements of cell molecules at the single cell resolution, has enabled to resolve the diversity of immune cell subsets, and their heterogeneous functionality. Here we assess the feasibility of taking CyTOF one step further to immuno profile cells while tracking their interactions with bacteria, a method we term Bac-CyTOF. We focus on the pathogen Klebsiella pneumoniae interrogating the pneumonia mouse model. Using Bac-CyTOF, we unveil the atlas of immune cells of mice infected with a K. pneumoniae hypervirulent strain. The atlas is characterized by a decrease in the populations of alveolar and monocyte-derived macrophages. Conversely, neutrophils, and inflammatory monocytes are characterized by an increase in the subpopulations expressing markers of less active cells such as the immune checkpoint PD-L1. These are the cells infected. We show that the type VI secretion system (T6SS) contributes to shape the lung immune landscape. The T6SS governs the interaction with monocytes/macrophages by shifting Klebsiella from alveolar macrophages to interstitial macrophages and limiting the infection of inflammatory monocytes. The lack of T6SS results in an increase of cells expressing markers of active cells, and a decrease in the subpopulations expressing PD-L1. By probing Klebsiella, and Acinetobacter baumannii strains with limited ability to survive in vivo, we uncover that a heightened recruitment of neutrophils, and relative high levels of alveolar macrophages and eosinophils and the recruitment of a characteristic subpopulation of neutrophils are features of mice clearing infections. We leverage Bac-CyTOF-generated knowledge platform to investigate the role of the DNA sensor STING in Klebsiella infections. sting-/- infected mice present features consistent with clearing the infection including the reduced levels of PD-L1. STING absence facilitates Klebsiella clearance.

Meeting report - Cell dynamics: host-pathogen interface.

Two years into the most significant infectious disease event of our generation, infections have populated every conversation and in-depth understanding of host-pathogen interactions has, perhaps, never been more important. In a successful return to in-person conferences, the host-pathogen interface was the focus of the third Cell Dynamics meeting, which took place at the glorious Wotton House in Surrey, UK. The meeting organised by Michaela Gack, Maximiliano Gutierrez, Dominique Soldati-Favre and Michael Way gathered an international group of scientists who shared their recent discoveries and views on numerous aspects, including cell-autonomous defence mechanisms, pathogen interactions with host cytoskeletal or membrane dynamics, and cellular immune regulation. More than 30 years into the beginning of cellular microbiology as a field, the meeting exhibited the unique aspect of the host-pathogen interface in uncovering the fundamentals of both pathogens and their hosts.

Visualisation of Host-Pathogen Communication.

The core of biomedical science is the use of laboratory techniques to support the diagnosis and treatment of disease in clinical settings. Despite tremendous advancement in our understanding of medicine in recent years, we are still far from having a complete understanding of human physiology in homeostasis, let alone the pathology of disease states. Indeed medical advances over the last two hundred years would not have been possible without the invention of and continuous development of visualisation techniques available to research scientists and clinicians. As we have all learned from the recent COVID pandemic, despite advances in modern medicine we still have much to learn regarding infection biology. Indeed antimicrobial resistant (AMR) bacteria are a global threat to human health, meaning research into bacterial pathogenesis is vital. In this chapter, we will briefly describe the nature of microbes and host immune responses before delving into some of the visualisation techniques utilised in the field of biomedical research with a focus on host-pathogen interactions. We will give a brief overview of commonly used techniques from gold standard staining methods, in situ hybridisation, microscopy, western blotting, microbial characterisation, to cutting-edge image flow cytometry and mass spectrometry. Specifically, we will focus on techniques utilised to visualise interactions between the host, our own bodies, and invading organisms including bacteria. We will touch on in vitro and ex vivo modelling methodology with examples utilised to delineate pathogenicity in disease. A better understanding of bacterial biology, immunology and how these fields interact (host-pathogen communications) in biomedical research is integral to developing novel therapeutic approaches which circumvent the need for antibiotics, an important issue as we enter a post-antibiotic era.

Laparoscopic colorectal surgery and discharge within 24 h-who is at risk for readmission?

AIM: The aim was to describe risk factors for hospital readmission in patients undergoing laparoscopic colorectal procedures and being discharged in ≤24 h. METHOD: All consecutive patients undergoing minimally invasive colorectal surgery between 2010 and 2019 from a single institution were retrospectively reviewed. All patients were included in an enhanced recovery programme. Patients who met criteria for hospital discharge were compared according to the need for readmission in a 45-day follow-up. RESULTS: In all, 664 patients underwent minimally invasive colorectal surgery during the study period and 237 (35.7%) were discharged in ≤24 h. Readmission was required in 16 (6.8%) patients discharged in ≤24 h and no postoperative mortality was observed in this group. Patients discharged in ≤24 h were more likely to have benign disease (P < 0.001), fewer associated procedures (P < 0.025) and intracorporeal anastomoses (P < 0.001). The type of surgical procedure (abdominoperineal resection), low rectal tumour, malignant disease, older age and longer operating time were associated with readmission. Age (OR 1.06; P = 0.037), malignant disease (OR 4.39; P = 0.05) and operating time (OR 1.03; P < 0.001) were identified as independent predictive factors for readmission amongst patients being discharged in ≤24 h. CONCLUSION: Highly selected patients undergoing minimally invasive procedures in colorectal surgery may be safely discharged within 24 h following the procedure. High-risk features for readmission include older age, malignant disease and longer operating time.

Burden of Disease Estimates of Seven Pathogens Commonly Transmitted Through Foods in Denmark, 2017.

Burden of disease metrics are increasingly established to prioritize food safety interventions. We estimated the burden of disease caused by seven foodborne pathogens in Denmark in 2017: Campylobacter, Salmonella, Shiga toxin-producing Escherichia coli, norovirus, Yersinia enterocolitica, Listeria monocytogenes, and Toxoplasma gondii. We used public health surveillance data and scientific literature to estimate incidence, mortality, and total disability-adjusted life year (DALY) of each, and linked results with estimates of the proportion of disease burden that is attributable to foods. Our estimates showed that Campylobacter caused the highest burden of disease, leading to a total burden of 1709 DALYs (95% uncertainty interval [UI] 1665-1755), more than threefold higher than the second highest ranked pathogen (Salmonella: 492 DALYs; 95% UI 481-504). Campylobacter still led the ranking when excluding DALYs attributable to nonfoodborne routes of exposure. The total estimated incidence was highest for norovirus, but this agent ranked sixth when focusing on foodborne burden. Salmonella ranked second in terms of foodborne burden of disease, followed by Listeria and Yersinia. Foodborne congenital toxoplasmosis was estimated to cause the loss of ∼100 years of healthy life, a burden that was borne by a low number of cases in the population. The ranking of foodborne pathogens varied substantially when based on reported cases, estimated incidence, and burden of disease estimates. Our results reinforce the need to continue food safety efforts throughout the food chain in Denmark, with a particular focus on reducing the incidence of Campylobacter infections.

2-Hydroxylation of Acinetobacter baumannii Lipid A Contributes to Virulence.

Acinetobacter baumannii causes a wide range of nosocomial infections. This pathogen is considered a threat to human health due to the increasingly frequent isolation of multidrug-resistant strains. There is a major gap in knowledge on the infection biology of A. baumannii, and only a few virulence factors have been characterized, including lipopolysaccharide. The lipid A expressed by A. baumannii is hepta-acylated and contains 2-hydroxylaurate. The late acyltransferases controlling the acylation of lipid A have been already characterized. Here, we report the characterization of A. baumannii LpxO, which encodes the enzyme responsible for the 2-hydroxylation of lipid A. By genetic methods and mass spectrometry, we demonstrate that LpxO catalyzes the 2-hydroxylation of the laurate transferred by A. baumannii LpxL. LpxO-dependent lipid A 2-hydroxylation protects A. baumannii from polymyxin B, colistin, and human ß-defensin 3. LpxO contributes to the survival of A. baumannii in human whole blood and is required for pathogen survival in the waxmoth Galleria mellonella LpxO also protects Acinetobacter from G. mellonella antimicrobial peptides and limits their expression. Further demonstrating the importance of LpxO-dependent modification in immune evasion, 2-hydroxylation of lipid A limits the activation of the mitogen-activated protein kinase Jun N-terminal protein kinase to attenuate inflammatory responses. In addition, LpxO-controlled lipid A modification mediates the production of the anti-inflammatory cytokine interleukin-10 (IL-10) via the activation of the transcriptional factor CREB. IL-10 in turn limits the production of inflammatory cytokines following A. baumannii infection. Altogether, our studies suggest that LpxO is a candidate for the development of anti-A. baumannii drugs.

Social behaviour and making attachments: a report from the fifth 'Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis'.

The fifth Young Microbiologists Symposium was held in Queen's University Belfast, Northern Ireland, in late August 2018. The symposium, focused on 'Microbe signalling, organization and pathogenesis', attracted 121 microbiologists from 15 countries. The meeting allowed junior scientists to present their work to a broad audience, and was supported by the European Molecular Biology Organization, the Federation of European Microbiological Societies, the Society of Applied Microbiology, the Biochemical Society and the Microbiology Society. Sessions covered recent advances in areas of microbiology including gene regulation and signalling, secretion and transport across membranes, infection and immunity, and antibiotics and resistance mechanisms. In this Meeting Report, we highlight some of the most significant advances and exciting developments communicated during talks and poster presentations.

The acetate uptake transporter family motif "NPAPLGL(M/S)" is essential for substrate uptake.

Organic acids are recognized as one of the most prevalent compounds in ecosystems, thus the transport and assimilation of these molecules represent an adaptive advantage for organisms. The AceTr family members are associated with the active transport of organic acids, namely acetate and succinate. The phylogenetic analysis shows this family is dispersed in the tree of life. However, in eukaryotes, it is almost limited to microbes, though reaching a prevalence close to 100% in fungi, with an essential role in spore development. Aiming at deepening the knowledge in this family, we studied the acetate permease AceP from Methanosarcina acetivorans, as the first functionally characterized archaeal member of this family. Furthermore, we demonstrate that the yeast Gpr1 from Yarrowia lipolytica is an acetate permease, whereas the Ady2 closest homologue in Saccharomyces cerevisiae, Fun34, has no role in acetate uptake. In this work, we describe the functional role of the AceTr conserved motif NPAPLGL(M/S). We further unveiled the role of the amino acid residues R122 and Q125 of SatP as essential for protein activity.

Yeast as a tool to express sugar acid transporters with biotechnological interest.

Sugar acids can be used as platform chemicals to generate primary building blocks of industrially relevant products. Microbial production of these organic compounds at high yields requires the engineering of the enzymatic machinery and the presence of plasma membrane transporters able to export them outside the cells. In this study, several yeast carboxylic acid transporters belonging to the Jen family were screened for the transport of biotechnologically relevant sugar acids, namely gluconic, saccharic, mucic, xylaric and xylonic acid, and functionally characterised in Saccharomyces cerevisiae. We show that Jen permeases are capable of transporting most of these sugar acids, although with different specificities. Saccharate is a substrate of the transporters ScJen1-S271Q and KlJen2, gluconate of CaJen2 and KlJen2, and xylarate and mucate of CaJen2. A molecular docking approach of these transporters identified the residues that play a major role in the substrate binding of these sugar acids, namely R188 (ScJen1), R122 (CaJen2) and R127 (KlJen2), all equivalent residues (TMS II). The identification of Jen members as sugar acid transporters can contribute to engineering efficient microbial cell factories with increased sugar acid production, as the ScJen1 is able to promote substrate efflux.

Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans.

AcpA has been previously characterized as a high-affinity transporter essential for the uptake and use of acetate as sole carbon source in Aspergillus nidulans. Here, we follow the expression profile of AcpA and define its substrate specificity. AcpA-mediated acetate transport is detected from the onset of conidiospore germination, peaks at the time of germ tube emergence, and drops to low basal levels in germlings and young mycelia, where a second acetate transporter is also becoming apparent. AcpA activity also responds to acetate presence in the growth medium, but is not subject to either carbon or nitrogen catabolite repression. Short-chain monocarboxylates (benzoate, formate, butyrate and propionate) inhibit AcpA-mediated acetate transport with apparent inhibition constants (Ki) of 16.89±2.12, 9.25±1.01, 12.06±3.29 and 1.44±0.13mM, respectively. AcpA is also shown not to be directly involved in ammonia export, as proposed for its Saccharomyces cerevisiae homologue Ady2p. In the second part of this work, we search for the unknown acetate transporter expressed in mycelia, and for other transporters that might contribute to acetate uptake. In silico analysis, genetic construction of relevant null mutants, and uptake assays, reveal that the closest AcpA homologue (AN1839), named AcpB, is the 'missing' secondary acetate transporter in mycelia. We also identify two major short-chain carboxylate (lactate, succinate, pyruvate and malate) transporters, named JenA (AN6095) and JenB (AN6703), which however are not involved in acetate uptake. This work establishes a framework for further exploiting acetate and carboxylate transport in filamentous ascomycetes.

SATP (YaaH), a succinate-acetate transporter protein in Escherichia coli.

In the present paper we describe a new carboxylic acid transporter in Escherichia coli encoded by the gene yaaH. In contrast to what had been described for other YaaH family members, the E. coli transporter is highly specific for acetic acid (a monocarboxylate) and for succinic acid (a dicarboxylate), with affinity constants at pH 6.0 of 1.24±0.13 mM for acetic acid and 1.18±0.10 mM for succinic acid. In glucose-grown cells the ΔyaaH mutant is compromised for the uptake of both labelled acetic and succinic acids. YaaH, together with ActP, described previously as an acetate transporter, affect the use of acetic acid as sole carbon and energy source. Both genes have to be deleted simultaneously to abolish acetate transport. The uptake of acetate and succinate was restored when yaaH was expressed in trans in ΔyaaH ΔactP cells. We also demonstrate the critical role of YaaH amino acid residues Leu¹³¹ and Ala¹64 on the enhanced ability to transport lactate. Owing to its functional role in acetate and succinate uptake we propose its assignment as SatP: the Succinate-Acetate Transporter Protein.

Description of patterns of ear and tail lesions during the grower-finisher period in a commercial pig farm.

BACKGROUND: Ear and tail lesions are prevalent indicators of impaired welfare observed in pig production with different multifactorial causes. Understanding the progression of ear and tail lesions over time is important to implement preventative strategies on commercial pig farms. Therefore, this case study aimed to provide a detailed account of patterns of ear and tail lesions in pigs on a single commercial farm during the grower-finisher period. CASE PRESENTATION: A total of 1,676 12-week old pigs (n = 773 females and n = 903 males, all tail docked) were followed from arrival to the grower facilities until transferred to the finisher stage on a commercial pig farm in Ireland. Pigs were individually weighed and inspected for the severity of fresh ear and tail lesions (score 0-4) on transfer to the first grower (24.9 ± 5.33 kg, 12 weeks of age, n = 1,676 pigs), second grower (33.3 ± 7.04 kg, 14 weeks of age, n = 1,641 pigs), and finisher stage (60.2 ± 7.74 kg, 18 weeks of age, n = 1,626 pigs). Due to the low number of pigs with high scores, ear lesions were classified as no (score 0), mild (score 1), moderate (score 2) and severe (score ≥ 3) and tail lesions were classified as no (score 0), mild (score 1), and moderate-to-severe (score ≥ 2). Ear lesions were more prevalent than tail lesions at each inspection. There were approx. 19% of pigs with ear lesions at all three inspections but no pigs presented with tail lesions at all three inspections. When considering the specific severity categories, we observed 32 different ear lesion score combinations and 15 different tail lesion score combinations across the three inspections. CONCLUSION: The high number of observed patterns of ear and tail lesions suggest large individual variability in lesion progression. Ear lesions were more of an issue than tail lesions and little is known about this health and welfare problem indicating that further research into causes and management strategies is needed.

The Role of Wildlife and Pests in the Transmission of Pathogenic Agents to Domestic Pigs: A Systematic Review.

Wild animals and pests are important reservoirs and vectors of pathogenic agents that can affect domestic pigs. Rapid globalization, anthropogenic factors, and increasing trends toward outdoor pig production facilitate the contact between domestic pigs and wildlife. However, knowledge on the transmission pathways between domestic pigs and the aforementioned target groups is limited. The present systematic review aims to collect and analyze information on the roles of different wild animal species and pests in the spread of pathogens to domesticated pigs. Overall, 1250 peer-reviewed manuscripts published in English between 2010 and 2022 were screened through the PRISMA framework using PubMed, Scopus, and Web of Science databases. A total of 84 studies reporting possible transmission routes of different pathogenic agents were included. A majority of the studies (80%) focused on the role of wild boars in the transmission of pathogenic agents to pig farms. Studies involving the role of rodents (7%), and deer (6%) were the next most frequent, whereas the role of insects (5%), wild carnivores (5%), wild birds (4%), cats (2%), and badgers (1%) were less available. Only 3.5% of studies presented evidence-based transmission routes from wildlife to domestic pigs. Approximately 65.5% of the included studies described possible risks/risk factors for pathogens' transmission based on quantitative data, whereas 31% of the articles only presented a hypothesis or qualitative analysis of possible transmission routes or risk factors and/or contact rates. Risk factors identified include outdoor farms or extensive systems and farms with a low level of biosecurity as well as wildlife behavior; environmental conditions; human activities and movements; fomites, feed (swill feeding), water, carcasses, and bedding materials. We recommend the strengthening of farm biosecurity frameworks with special attention to wildlife-associated parameters, especially in extensive rearing systems and high-risk zones as it was repeatedly found to be an important measure to prevent pathogen transmission to domestic pigs. In addition, there is a need to focus on effective risk-based wildlife surveillance mechanisms and to raise awareness among farmers about existing wildlife-associated risk factors for disease transmission.

Value of simplified lung lesions scoring systems to inform future codes for routine meat inspection in pigs.

BACKGROUND: Across the European Union (EU), efforts are being made to achieve modernisation and harmonisation of meat inspection (MI) code systems. Lung lesions were prioritised as important animal based measures at slaughter, but existing standardized protocols are difficult to implement for routine MI. This study aimed to compare the informative value and feasibility of simplified lung lesion scoring systems to inform future codes for routine post mortem MI. RESULTS: Data on lung lesions in finisher pigs were collected at slaughter targeting 83 Irish pig farms, with 201 batches assessed, comprising 31,655 pairs of lungs. Lungs were scored for cranioventral pulmonary consolidations (CVPC) and pleurisy lesions using detailed scoring systems, which were considered the gold standard. Using the data collected, scenarios for possible simplified scoring systems to record CVPC (n = 4) and pleurisy (n = 4) lesions were defined. The measurable outcomes were the prevalence and (if possible) severity scoring at batch level for CVPC and pleurisy. An arbitrary threshold was set to the upper quartile (i.e., the top 25% of batches with high prevalence/severity of CVPC or pleurisy, n = 50). Each pair of measurable outcomes was compared by calculating Spearman rank correlations and assessing if batches above the threshold for one measurable outcome were also above it for their pairwise comparison. All scenarios showed perfect agreement (k = 1) when compared among themselves and the gold standard for the prevalence of CVPC. The agreement among severity outcomes and the gold standard showed moderate to perfect agreement (k = [0.66, 1]). The changes in ranking were negligible for all measurable outcomes of pleurisy for scenarios 1, 2 and 3 when compared with the gold standard (rs ≥ 0.98), but these changes amounted to 50% for scenario 4. CONCLUSIONS: The best simplified CVPC scoring system is to simply count the number of lung lobes affected excluding the intermediate lobe, which provides the best trade-off between value of information and feasibility, by incorporating information on CVPC prevalence and severity. While for pleurisy evaluation, scenario 3 is recommended. This simplified scoring system provides information on the prevalence of cranial and moderate and severe dorsocaudal pleurisy. Further validation of the scoring systems at slaughter and by private veterinarians and farmers is needed.

Modelling the Gastrointestinal Carriage of Klebsiella pneumoniae Infections.

Klebsiella pneumoniae is a leading cause of nosocomial and community acquired infections, making K. pneumoniae the pathogen that is associated with the second largest number of deaths attributed to any antibiotic resistant infection. K. pneumoniae colonizes the nasopharynx and the gastrointestinal tract in an asymptomatic manner without dissemination to other tissues. Importantly, gastrointestinal colonization is a requisite for infection. Our understanding of K. pneumoniae colonization is still based on interrogating mouse models in which animals are pretreated with antibiotics to disturb the colonization resistance imposed by the gut microbiome. In these models, infections disseminate to other tissues. Here, we report a murine model to allow for the study of the gastrointestinal colonization of K. pneumoniae without tissue dissemination. Hypervirulent and antibiotic resistant strains stably colonize the gastrointestinal tract of in an inbred mouse population without antibiotic treatment. The small intestine is the primary site of colonization and is followed by a transition to the colon over time, without dissemination to other tissues. Our model recapitulates the disease dynamics of the metastatic K. pneumoniae strains that are able to disseminate from the gastrointestinal tract to other sterile sites. Colonization is associated with mild to moderate histopathology, no significant inflammation, and no effect on the richness of the microbiome. Our model sums up the clinical scenario in which antibiotic treatment disturbs the colonization of K. pneumoniae and results in dissemination to other tissues. Finally, we establish that the capsule polysaccharide is necessary for the colonization of the large intestine, whereas the type VI secretion system contributes to colonization across the gastrointestinal tract. IMPORTANCE Klebsiella pneumoniae is one of the pathogens that is sweeping the world in the antibiotic resistance pandemic. Klebsiella colonizes the nasopharynx and the gut of healthy subjects in an asymptomatic manner, making gut colonization a requisite for infection. This makes it essential to understand the gastrointestinal carriage in preventing Klebsiella infections. Current research models rely on the perturbation of the gut microbiome by antibiotics, resulting in an invasive infection. Here, we report a new model of K. pneumoniae gut colonization that recapitulates key features of the asymptomatic human gastrointestinal tract colonization. In our model, there is no need to disturb the microbiota to achieve stable colonization, and there is no dissemination to other tissues. Our model sums up the clinical scenario in which antibiotic treatment triggers invasive infection. We envision that our model will be an excellent platform upon which to investigate factors enhancing colonization and invasive infections and to test therapeutics to eliminate Klebsiella asymptomatic colonization.

A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection.

Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFß-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.

PYHIN protein IFI207 regulates cytokine transcription and IRF7 and contributes to the establishment of K. pneumoniae infection.

PYHIN proteins AIM2 and IFI204 sense pathogen DNA, while other PYHINs have been shown to regulate host gene expression through as-yet unclear mechanisms. We characterize mouse PYHIN IFI207, which we find is not involved in DNA sensing but rather is required for cytokine promoter induction in macrophages. IFI207 co-localizes with both active RNA polymerase II (RNA Pol II) and IRF7 in the nucleus and enhances IRF7-dependent gene promoter induction. Generation of Ifi207-/- mice shows no role for IFI207 in autoimmunity. Rather, IFI207 is required for the establishment of a Klebsiella pneumoniae lung infection and for Klebsiella macrophage phagocytosis. These insights into IFI207 function illustrate that PYHINs can have distinct roles in innate immunity independent of DNA sensing and highlight the need to better characterize the whole mouse locus, one gene at a time.

A substrate translocation trajectory in a cytoplasm-facing topological model of the monocarboxylate/H⁺ symporter Jen1p.

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H⁺ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence ³79NXX[S/T]HX[S/T]QD³87 in transmembrane segment VII (TMS-VII) is part of the substrate translocation pathway. Here, we rationally design, analyse and show that several novel mutations in TMS-V and TMS-XI directly modify Jen1p function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are critical specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is a critical residue for function. Using a model created on the basis of Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI (N379, H383, D387, Q498, N501) are perfectly aligned in an imaginary axis that lies parallel to the protein pore. This model and subsequent mutational analysis further reveal that an additional polar residue facing the pore, R188 (TMS-II), is irreplaceable for function. Our model also justifies the role of F270 and Q498 in substrate specificity. Finally, docking calculations reveal a 'trajectory-like' substrate displacement within the Jen1p pore, where R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.

Lactic acid production in Saccharomyces cerevisiae is modulated by expression of the monocarboxylate transporters Jen1 and Ady2.

We aimed to manipulate the metabolism of Saccharomyces cerevisiae to produce lactic acid and search for the potential influence of acid transport across the plasma membrane in this process. Saccharomyces cerevisiae W303-1A is able to use l-lactic acid but its production in our laboratory has not previously been detected. When the l-LDH gene from Lactobacillus casei was expressed in S. cerevisiae W303-1A and in the isogenic mutants jen1∆, ady2∆ and jen1∆ ady2∆, all strains were able to produce lactic acid, but higher titres were achieved in the mutant strains. In strains constitutively expressing both LDH and JEN1 or ADY2, a higher external lactic acid concentration was found when glucose was present in the medium, but when glucose was exhausted, its consumption was more pronounced. These results demonstrate that expression of monocarboxylate permeases influences lactic acid production. Ady2 has been previously characterized as an acetate permease but our results demonstrated its additional role in lactate uptake. Overall, we demonstrate that monocarboxylate transporters Jen1 and Ady2 are modulators of lactic acid production and may well be used to manipulate lactic acid export in yeast cells.

Effect of Phase Feeding, Space Allowance and Mixing on Productive Performance of Grower-Finisher Pigs.

This study investigates the effects of space allowance (SA), mixing and phase feeding (PF) on performance of grower-finisher pigs. Three trials (T) were conducted. In T1 and T2, 345 pigs/trial were moved to finisher stage at 11 weeks of age and assigned to two SAs: 0.96 (n = 15 pens; 10 pigs/pen) and 0.78 (n = 15; 13 pigs/pen) m2/pig. Mixing was applied to 5 pens of each SA leading to a 2 × 2 factorial arrangement (SA × Mixing). For PF, 2 diets with 0.95 and 0.82 g SID Lys/MJ NE were applied to 5 pens of each SA (not mixed) leading to another 2 × 2 factorial arrangement (SA × PF). In T3, 230 pigs were moved to the grower-finisher stage at 11 weeks of age, mixed, and assigned to 4 treatments (SA × PF; n = 5 pens). Data were analyzed using general linear mixed models. SA did not affect performance (p > 0.05). Non-mixed pigs were 5.40 (T1) and 5.25 (T2) kg heavier than mixed pigs at 21 weeks of age (p < 0.001). PF reduced performance of pigs by 3.45 (T1) and 4.05 (T2) kg at 21 weeks of age (p < 0.001). In conclusion, mixing and reducing SID Lys:NE ratio from 0.95 to 0.82 g/MJ at 15-16 weeks of age, have a more marked impact on performance than reducing SA from 0.96 to 0.78 m2/pig.