| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 3. |
- Castanheira, Sonia, et al.
(författare)
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A Specialized Peptidoglycan Synthase Promotes Salmonella Cell Division inside Host Cells
- 2017
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Ingår i: mBio. - 2161-2129 .- 2150-7511. ; 8:6
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial cell division has been studied extensively under laboratory conditions. Despite being a key event in the bacterial cell cycle, cell division has not been explored in vivo in bacterial pathogens interacting with their hosts. We discovered in Salmonella enterica serovar Typhimurium a gene absent in nonpathogenic bacteria and encoding a peptidoglycan synthase with 63% identity to penicillin-binding protein 3 (PBP3). PBP3 is an essential cell division-specific peptidoglycan synthase that builds the septum required to separate daughter cells. Since S. Typhimurium carries genes that encode a PBP3 paralog-which we named PBP3(SAL)-and PBP3, we hypothesized that there are different cell division events in host and nonhost environments. To test this, we generated S. Typhimurium isogenic mutants lacking PBP3(SAL) or the hitherto considered essential PBP3. While PBP3 alone promotes cell division under all conditions tested, the mutant producing only PBP3(SAL) proliferates under acidic conditions (pH <= 5.8) but does not divide at neutral pH. PBP3(SAL) production is tightly regulated with increased levels as bacteria grow in media acidified up to pH 4.0 and in intracellular bacteria infecting eukaryotic cells. PBP3(SAL) activity is also strictly dependent on acidic pH, as shown by beta-lactam antibiotic binding assays. Live-cell imaging microscopy revealed that PBP3(SAL) alone is sufficient for S. Typhimurium to divide within phagosomes of the eukaryotic cell. Additionally, we detected much larger amounts of PBP3(SAL) than those of PBP3 in vivo in bacteria colonizing mouse target organs. Therefore, PBP3(SAL) evolved in S. Typhimurium as a specialized peptidoglycan synthase promoting cell division in the acidic intraphagosomal environment. IMPORTANCE During bacterial cell division, daughter cells separate by a transversal structure known as the division septum. The septum is a continuum of the cell wall and therefore is composed of membrane(s) and a peptidoglycan layer. To date, actively growing bacteria were reported to have only a "cell division-specific" peptidoglycan synthase required for the last steps of septum formation and consequently, essential for bacterial life. Here, we discovered that Salmonella enterica has two peptidoglycan synthases capable of synthesizing the division septum. One of these enzymes, PBP3(SAL), is present only in bacterial pathogens and evolved in Salmonella to function exclusively in acidic environments. PBP3(SAL) is used preferentially by Salmonella to promote cell division in vivo in mouse target organs and inside acidified phagosomes. Our data challenge the concept of only one essential cell division-specific peptidoglycan synthase and demonstrate that pathogens can divide in defined host locations using alternative mechanisms.
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| 4. |
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| 5. |
- de Oliveira, Ana Henriques, et al.
(författare)
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Listeria monocytogenes requires the RsbX protein to prevent SigB-activation under non-stressed conditions
- 2022
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 204:1
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Tidskriftsartikel (refereegranskat)abstract
- The survival of microbial cells under changing environmental conditions requires an efficient reprogramming of transcription, often mediated by alternative sigma factors. The Gram-positive human pathogen Listeria monocytogenes senses and responds to environmental stress mainly through the alternative sigma factor σB (SigB), which controls expression of the general stress response regulon. SigB activation is achieved through a complex series of phosphorylation/dephosphorylation events culminating in the release of SigB from its anti-sigma factor RsbW. At the top of the signal transduction pathway lies a large multi-protein complex known as the stressosome that is believed to act as a sensory hub for stresses. Following signal detection, stressosome proteins become phosphorylated. Resetting of the stressosome is hypothesized to be exerted by a putative phosphatase, RsbX, which presumably removes phosphate groups from stressosome proteins post-stress.We addressed the role of the RsbX protein in modulating the activity of the stressosome and consequently regulating SigB activity in L. monocytogenes. We show that RsbX is required to reduce SigB activation/levels under non-stress conditions and that it is required for appropriate SigB mediated stress-adaptation. A strain lacking RsbX displayed impaired motility and biofilm formation, but also an increased survival at low pH. Our results could suggest that absence of RsbX alter the multi-protein composition of the stressosome without dramatically affecting its phosphorylation status. Overall the data show that RsbX plays a critical role in modulating the signal transduction pathway by blocking SigB activation under non-stressed conditions.
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| 6. |
- de Oliveira, Ana H., 1995-, et al.
(författare)
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The virulence and infectivity of Listeria monocytogenes are not substantially altered by elevated SigB activity
- 2023
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 91:6
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Tidskriftsartikel (refereegranskat)abstract
- Listeria monocytogenes is a bacterial pathogen capable of causing severe infections but also thriving outside the host. To respond to different stress conditions, L. monocytogenes mainly utilizes the general stress response regulon, which largely is controlled by the alternative sigma factor Sigma B (SigB). In addition, SigB is important for virulence gene expression and infectivity. Upon encountering stress, a large multicomponent protein complex known as the stressosome becomes activated, ultimately leading to SigB activation. RsbX is a protein needed to reset a "stressed"stressosome and prevent unnecessary SigB activation in nonstressed conditions. Consequently, absence of RsbX leads to constitutive activation of SigB even without prevailing stress stimulus. To further examine the involvement of SigB in the virulence of this pathogen, we investigated whether a strain with constitutively active SigB would be affected in virulence factor expression and/or infectivity in cultured cells and in a chicken embryo infection model. Our results suggest that increased SigB activity does not substantially alter virulence gene expression compared with the wild-type (WT) strain at transcript and protein levels. Bacteria lacking RsbX were taken up by phagocytic and nonphagocytic cells at a similar frequency to WT bacteria, both in stressed and nonstressed conditions. Finally, the absence of RsbX only marginally affected the ability of bacteria to infect chicken embryos. Our results suggest only a minor role of RsbX in controlling virulence factor expression and infectivity under these conditions.
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| 7. |
- Demuth, Andreas, et al.
(författare)
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Pathogenomics: An updated European Research Agenda
- 2008
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Ingår i: Infection, Genetics and Evolution. - : Elsevier BV. - 1567-7257 .- 1567-1348. ; 8:3, s. 386-393
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Tidskriftsartikel (refereegranskat)abstract
- The emerging genomic technologies and bioinformatics provide novel opportunities for studying life-threatening human pathogens and to develop new applications for the improvement of human and animal health and the prevention, treatment, and diagnosis of infections. Based on the ecology and population biology of pathogens and related organisms and their connection to epidemiology, more accurate typing technologies and approaches will lead to better means of disease control. The analysis of the genome plasticity and gene pools of pathogenic bacteria including antigenic diversity and antigenic variation results in more effective vaccines and vaccine implementation programs. The study of newly identified and uncultivated microorganisms enables the identification of new threats. The scrutiny of the metabolism of the pathogen in the host allows the identification of new targets for anti-infectives and therapeutic approaches. The development of modulators of host responses and mediators of host damage will be facilitated by the research on interactions of microbes and hosts, including mechanisms of host damage, acute and chronic relationships as well as commensalisms. The study of multiple pathogenic and non-pathogenic microbes interacting in the host will improve the management of multiple infections and will allow probiotic and prebiotic interventions. Needless to iterate, the application of the results of improved prevention and treatment of infections into clinical tests will have a positive impact on the management of human and animal disease. The Pathogenomics Research Agenda draws on discussions with experts of the Network of Excellence "EuroPathoGenomics" at the management board meeting of the project held during 18-21 April 2007, in the Villa Vigoni, Menaggio, Italy. Based on a proposed European Research Agenda in the field of pathogenomics by the ERA-NET PathoGenoMics the meeting's participants updated the established list of topics as the research agenda for the future.
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| 8. |
- Ek, Viktor
(författare)
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It Takes Two to Tango : Bacterial heterogeneity and host cell features govern Salmonella infection
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Salmonella enterica serovar Typhimurium (S.Tm) causes enterocolitis with significant worldwide morbidity and mortality. The general aim of this thesis is to investigate variation in host cell invasion mechanisms used by S.Tm across different host cell contexts, as well as the influence of bacterial cell-cell heterogeneity on invasion-relevant S.Tm behaviours. The thesis is divided into four sub-projects, each a section in the presented work.First, a genetic barcoding technique for tracking bacteria in mixed consortium infections was developed and applied to evaluate the dependency on the type three secretion system 1 (T3SS-1) and its effectors for host cell entry. It was found that S.Tm invasion of cultured epithelial cells and monocytes is mainly mediated by T3SS-1, or by cooperative uptake of bystander bacteria. T3SS-1-independent entry was possible in cultured macrophages, although T3SS-1-dependent entry was predominant also there. In fact, active invasion was promoted by the same T3SS-1 effectors in all three cell types. Second, an in-depth comparison of S.Tm infections in cell line cultures and in the mouse gut mucosa in vivo highlighted a “discreet-invasion” modality in vivo, in sharp contrast to the prevailing “ruffle” model for host cell invasion. While ruffle-mediated entry into epithelial cell lines was driven by the T3SS-1 effectors SopBEE2, discreet-invasion into the murine gut absorptive epithelium is driven predominantly by the SipA effector, as well as the SiiE adhesin. Furthermore, discreet-invasion targeted apicolateral “hot spots” near cell-cell junctions, dependent on the local cell neighbourhood, which was further charted in the final two sub-projects. Third, single-bacterium characteristics among S.Tm populations were studied using time-lapse microscopy. The indistinct nature of the shift from growth to virulence induction spawned a transient subpopulation of S.Tm “doublets”, cell division intermediates also exhibiting pronounced swimming and host cell invasion aptitude. The longer doublets also displayed a different search pattern during near-surface swimming, highlighting bacterial cell length heterogeneity as a key determinant of target search atop epithelia. Fourth, the morphogenic impact of clinically relevant antibiotics were explored, in context of the previous data. Even S.Tm bacteria with the most extreme morphological abnormalities (e.g. highly filamentous or coccoid individuals), induced by chloramphenicol, ciprofloxacin, nitrofurantoin, and meropenem, could robustly swim and invade epithelial host cells. While high concentrations of these antibiotics were effective at suppressing growth and virulence, a range of low, sub-inhibitory concentrations even enhanced host cell invasion capacity and affected the near-surface swimming behaviour among surviving bacteria. In summary, the present investigation highlights the pivotal importance of taking both host cell features and bacterial heterogeneity into account when studying infection processes.
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| 9. |
- Guerreiro, Duarte N., et al.
(författare)
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Acid stress signals are integrated into the σb-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes
- 2022
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Ingår i: PLoS Pathogens. - : Public Library of Science. - 1553-7366 .- 1553-7374. ; 18:3
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Tidskriftsartikel (refereegranskat)abstract
- The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B (σB), whose role in protection against acid stress is well established. Here, we investigated the involvement of the stressosome, a sensory hub, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated σB and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was solely present retained the ability to induce σB activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1-T175A,-T209A,-T241A) and RsbS (rsbS-S56A), or the stressosome kinase RsbT (rsbTN49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1-T209A and rsbR1-T241A mutants displayed constitutive σB activity. Mild acid shock upregulates invasion genes inlAB and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of σB in L. monocytogenes. Furthermore, they show that RsbR1 can function independently of its paralogues and signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209 but not T175. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.
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| 10. |
- Guerreiro, Duarte N., et al.
(författare)
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Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes
- 2020
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 202:9
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Tidskriftsartikel (refereegranskat)abstract
- In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype.IMPORTANCE: In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.
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