| 1. |
- Avican, Kemal, et al.
(author)
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Reprogramming of Yersinia from Virulent to Persistent Mode Revealed by Complex In Vivo RNA-seq Analysis
- 2015
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 11:1
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Journal article (peer-reviewed)abstract
- We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.
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| 2. |
- Bachmann, Julie, et al.
(author)
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Affinity Proteomics Reveals Elevated Muscle Proteins in Plasma of Children with Cerebral Malaria
- 2014
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 10:4, s. e1004038-
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Journal article (peer-reviewed)abstract
- Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.
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| 3. |
- Bollampalli, V. P., et al.
(author)
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BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming
- 2015
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In: PLoS Pathogens. - : Public Library of Science. - 1553-7366 .- 1553-7374. ; 11:10
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Journal article (peer-reviewed)abstract
- The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.
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| 4. |
- Bradley, Frideborg, et al.
(author)
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Multi-omics analysis of the cervical epithelial integrity of women using depot medroxyprogesterone acetate
- 2022
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 18:5
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Journal article (peer-reviewed)abstract
- Depot medroxyprogesterone acetate (DMPA) is an injectable hormonal contraceptive used by millions of women worldwide. However, experimental studies have associated DMPA use with genital epithelial barrier disruption and mucosal influx of human immunodeficiency virus (HIV) target cells. We explored the underlying molecular mechanisms of these findings. Ectocervical biopsies and cervicovaginal lavage (CVL) specimens were collected from HIV-seronegative Kenyan sex workers using DMPA (n = 32) or regularly cycling controls (n = 64). Tissue samples were assessed by RNA-sequencing and quantitative imaging analysis, whereas protein levels were measured in CVL samples. The results suggested a DMPA-associated upregulation of genes involved in immune regulation, including genes associated with cytokine-mediated signaling and neutrophil-mediated immunity. A transcription factor analysis further revealed DMPA-associated upregulation of RELA and NFKB1 which are involved in several immune activation pathways. Several genes significantly downregulated in the DMPA versus the control group were involved in epithelial structure and function, including genes encoding keratins, small proline-rich proteins, and cell-cell adhesion proteins. Pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development, including keratinization and cornification processes. The cervicovaginal microbiome composition (Lactobacillus dominant and non-Lactobacillus dominant) had no overall interactional impact on the DMPA associated tissue gene expression. Imaging analysis verified that DMPA use was associated with an impaired epithelial layer as illustrated by staining for the selected epithelial junction proteins E-cadherin, desmoglein-1 and claudin-1. Additional staining for CD4(+) cells revealed a more superficial location of these cells in the ectocervical epithelium of DMPA users versus controls. Altered protein levels of SERPINB1 and ITIH2 were further observed in the DMPA group. Identification of specific impaired epithelial barrier structures at the gene expression level, which were verified at the functional level by tissue imaging analysis, illustrates mechanisms by which DMPA adversely may affect the integrity of the genital mucosa. Author summarySexual transmission accounts for the majority of all new HIV infections in women, and alterations to the mucosal environment of the female genital tract have been associated with an increase in the risk of acquiring HIV. Observational epidemiological studies have implied that the use of the injectable hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may be associated with increased HIV-acquisition. However, a prospective clinical study has not confirmed this association and the controversial findings are currently evaluated in the context of international reproductive health policies. Several studies using various model systems indicate that DMPA affects the integrity of the genital epithelial barrier as well as the mucosal immune system, but the exact mechanisms remain largely unknown. To characterize the effect of DMPA on the genital mucosal environment, we used a multi-omics approach to assess paired genital secretions and cervical tissue samples from long-term regular DMPA users living in Kenya. This unique cohort represents a population at risk of HIV infection in which DMPA is one of the most commonly used hormonal contraceptives. We identified impaired cervical epithelial barrier structures, including DMPA-associated reduction in the expression of cell-cell adhesion molecules, keratins, small proline-rich proteins and a thinner upper epithelial layer with more superficially located CD4(+) cells. Gene set enrichment pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development including keratinization and cornification pathways. Protein analysis identified altered levels of selected anti-proteases. Our findings illustrate mechanisms by which DMPA adversely may affect the integrity of the genital mucosa.
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| 5. |
- Duru, Adil Doganay, et al.
(author)
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Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination
- 2020
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In: PLoS Pathogens. - : PUBLIC LIBRARY SCIENCE. - 1553-7366 .- 1553-7374. ; 16:5
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Journal article (peer-reviewed)abstract
- Viral escape from CD8(+) cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8(+) T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape. Author summary Viral escape mutagenesis correlates often with disease progression and represents a major hurdle for vaccination-based therapies. Here, we have designed and developed a novel generation of altered epitopes that re-establish and enhance significantly CD8(+) T cell recognition of a naturally occurring viral immune escape variant. Biophysical and structural analyses provide a clear understanding of the molecular mechanisms underlying this reestablished recognition. We believe that this approach can be implemented to currently available or novel vaccination approaches to efficiently restore T cell recognition of virus escape variants to control disease progression.
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| 6. |
- Guerriero, Gea, et al.
(author)
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Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs
- 2010
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In: PLOS PATHOG. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 6:8, s. e1001070-
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Journal article (peer-reviewed)abstract
- Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.
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| 7. |
- Parameswaran, Poornima, et al.
(author)
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Six RNA Viruses and Forty-One Hosts : Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems
- 2010
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 6:2, s. e1000764-
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Journal article (peer-reviewed)abstract
- We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare'' (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs''). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 39 overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.
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| 8. |
- Powers, TuShun R., et al.
(author)
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Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica
- 2021
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:8, s. e1009280-
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Journal article (peer-reviewed)abstract
- Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.
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| 9. |
- Steiner, Svava E., et al.
(author)
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UPEC kidney infection triggers neuro-immune communication leading to modulation of local renal inflammation by splenic IFNγ
- 2021
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:5
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Journal article (peer-reviewed)abstract
- Bacterial infection results in a veritable cascade of host responses, both local and systemic. To study the initial stages of host-pathogen interaction in living tissue we use spatially-temporally controlled in vivo models. Using this approach, we show here that within 4 h of a uropathogenic Escherichia coli (UPEC) infection in the kidney, an IFNγ response is triggered in the spleen. This rapid infection-mediated inter-organ communication was found to be transmitted via nerve signalling. Bacterial expression of the toxin α-hemolysin directly and indirectly activated sensory neurons, which were identified in the basement membrane of renal tubules. Nerve activation was transmitted via the splenic nerve, inducing upregulation of IFNγ in the marginal zones of the spleen that led to increasing concentrations of IFNγ in the circulation. We found that IFNγ modulated the inflammatory signalling generated by renal epithelia cells in response to UPEC infection. This demonstrates a new concept in the host response to kidney infection; the role of nerves in sensing infection and rapidly triggering a systemic response which can modulate inflammation at the site of infection. The interplay between the nervous and immune systems is an exciting, developing field with the appealing prospect of non-pharmaceutical interventions. Our study identifies an important role for systemic neuro-immune communication in modulating inflammation during the very first hours of a local bacterial infection in vivo.
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| 10. |
- Tabusi, Mahebali, et al.
(author)
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Neuronal death in pneumococcal meningitis is triggered by pneumolysin and RrgA interactions with beta-actin
- 2021
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:3
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Journal article (peer-reviewed)abstract
- Neuronal damage is a major consequence of bacterial meningitis, but little is known about mechanisms of bacterial interaction with neurons leading to neuronal cell death. Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis and many survivors develop neurological sequelae after the acute infection has resolved, possibly due to neuronal damage. Here, we studied mechanisms for pneumococcal interactions with neurons. Using human primary neurons, pull-down experiments and mass spectrometry, we show that pneumococci interact with the cytoskeleton protein beta-actin through the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply), thereby promoting adhesion and invasion of neurons, and neuronal death. Using our bacteremia-derived meningitis mouse model, we observe that RrgA- and Ply-expressing pneumococci co-localize with neuronal beta-actin. Using purified proteins, we show that Ply, through its cholesterol-binding domain 4, interacts with the neuronal plasma membrane, thereby increasing the exposure on the outer surface of beta-actin filaments, leading to more beta-actin binding sites available for RrgA binding, and thus enhanced pneumococcal interactions with neurons. Pneumococcal infection promotes neuronal death possibly due to increased intracellular Ca2+ levels depending on presence of Ply, as well as on actin cytoskeleton disassembly. STED super-resolution microscopy showed disruption of beta-actin filaments in neurons infected with pneumococci expressing RrgA and Ply. Finally, neuronal death caused by pneumococcal infection could be inhibited using antibodies against beta-actin. The generated data potentially helps explaining mechanisms for why pneumococci frequently cause neurological sequelae. Author summary Neuronal damage is a major consequence of meningitis. Streptococcus pneumoniae (pneumococcus) is the leading etiological cause of bacterial meningitis, yet how pneumococci interact with neurons and cause neuronal death is poorly understood. Using human neurons in vitro and our established bacteremia-derived meningitis mouse model in vivo, we found that pneumococci use the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply) to interact with neuronal beta-actin expressed on the plasma membrane. Also, we demonstrate that Ply interaction with the neuronal plasma membrane increase the exposure of beta-actin on the neuronal plasma membrane, allowing more pneumococci to adhere to neurons through RrgA-beta-actin interaction. Moreover, neurons infected with RrgA- and Ply-expressing pneumococci showed increased intracellular Ca2+ levels and disruption of beta-actin filaments, possibly leading to neuronal death. Importantly, by blocking pneumococcal-beta-actin interaction using antibodies, we could reduce neuronal cell death after pneumococcal infection.
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