Phagocytosis resistance is an important virulence factor in Klebsiella pneumoniae. Dictyostelium has been used to study the interaction between phagocytes and bacteria because of its similarity to ...mammalian macrophages. In this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2,500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by DICTYOSTELIUM: In the 29 mutants, six loci were identified; three were capsular synthesis genes. Of the other three, one was related to carnitine metabolism, one encoded a subunit of protease (clpX), and one encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only ΔclpX and Δwzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of ΔclpX was examined by microarray analysis and revealed a 3-fold lower level of expression of capsular synthesis genes. Therefore, we have identified genes involved in resistance to phagocytosis in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily encapsulated bacteria.
Sweetening the deal: N. meningitidis serogroup W135 capsular oligosaccharides were synthesized in lengths from disaccharides to decasaccharides. Sera from mice immunized with these ...oligosaccharide–protein conjugates were examined by a glycan microarray (see picture) and bactericidal assay for antibody specificity and the ability to kill bacteria.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
•An lightweight ResNet-41 model is proposed for kidney segmentation in abdomen CT images.•An optimized kidney volume calculation system is proposed to determine the kidney volume precisely.•ResNet-41 ...and EfficientNet deep learning models are evaluated on the KiTS19 database and achieved a Dice score of 0.9648 for kidney segmentation and 0.7294 for all types of tumor detections, respectively.
Previously, doctors interpreted computed tomography (CT) images based on their experience in diagnosing kidney diseases. However, with the rapid increase in CT images, such interpretations were required considerable time and effort, producing inconsistent results. Several novel neural network models were proposed to automatically identify kidney or tumor areas in CT images for solving this problem. In most of these models, only the neural network structure was modified to improve accuracy. However, data pre-processing was also a crucial step in improving the results. This study systematically discussed the necessary pre-processing methods before processing medical images in a neural network model. The experimental results were shown that the proposed pre-processing methods or models significantly improve the accuracy rate compared with the case without data pre-processing. Specifically, the dice score was improved from 0.9436 to 0.9648 for kidney segmentation and 0.7294 for all types of tumor detections. The performance was suitable for clinical applications with lower computational resources based on the proposed medical image processing methods and deep learning models. The cost efficiency and effectiveness were also achieved for automatic kidney volume calculation and tumor detection accurately.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are ...critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K(1) (-) O(1)) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
Streptococcus pneumoniae
is a common cause of post-influenza secondary bacterial infection, which results in excessive morbidity and mortality. Although 13-valent pneumococcal ...conjugate vaccine (PCV13) vaccination programs have decreased the incidence of pneumococcal pneumonia, PCV13 failed to prevent serotype 3 pneumococcal disease as effectively as other vaccine serotypes. We aimed to investigate the mechanisms underlying the co-pathogenesis of influenza virus and serotype 3 pneumococci.
Methods
We carried out a genome-wide screening of a serotype 3
S. pneumoniae
transposon insertion mutant library in a mouse model of coinfection with influenza A virus (IAV) to identify the bacterial factors required for this synergism.
Results
Direct, high-throughput sequencing of transposon insertion sites identified 24 genes required for both coinfection and bacterial infection alone. Targeted deletion of the putative aminotransferase (
PA
) gene decreased bacterial growth, which was restored by supplementation with methionine. The bacterial burden in a coinfection with the
PA
gene deletion mutant and IAV in the lung was lower than that in a coinfection with wild-type pneumococcus and IAV, but was significantly higher than that in an infection with the
PA
gene deletion mutant alone. These data suggest that IAV infection alters host metabolism to benefit pneumococcal fitness and confer higher susceptibility to pneumococcal infection. We further demonstrated that bacterial growth was increased by supplementation with methionine or IAV-infected mouse lung homogenates.
Conclusions
The data indicates that modulation of host metabolism during IAV infection may serve as a potential therapeutic intervention against secondary bacterial infections caused by serotype 3 pneumococci during IAV outbreaks in the future.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Analysis of the genome of Klebsiella pneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with ...CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5'-TTN-3' was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Mucoviscosity-associated gene A (magA) of Klebsiella pneumoniae contributes to K1 capsular polysaccharide (CPS) biosynthesis. Based on sequence homology and gene alignment, the magA gene has been ...predicted to encode a Wzy-type CPS polymerase. Sequence alignment with the Wzy_C and RfaL protein families (which catalyze CPS or lipopolysaccharide (LPS) biosynthesis) and topological analysis has suggested that eight highly conserved residues, including G308, G310, G334, G337, R290, P305, H323, and N324, were located in a hypothetical loop region. Therefore, we used site-directed mutagenesis to study the role of these residues in CPS production, and to observe the consequent phenotypes such as mucoviscosity, serum and phagocytosis resistance, and virulence (as assessed in mice) in pyogenic liver abscess strain NTUH-K2044. Alanine substitutions at R290 or H323 abolished all of these properties. The G308A mutant was severely impaired for these functions. The G334A mutant remained mucoid with decreased CPS production, but its virulence was significantly reduced in vivo. No phenotypic change was observed for strains harboring magA G310A, G337A, P305A, or N324A mutations. Therefore, R290, G308, H323, and G334 are functionally important residues of the MagA (Wzy) protein of K. pneumoniae NTUH-K2044, capsular type K1. These amino acids are also likely to be important for the function of Wzy in other capsular types in K. pneumoniae and other species bearing Wzy_C family proteins.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Tuberculosis (TB) is an infectious respiratory disease caused by
Mycobacterium tuberculosis
and one of the top 10 causes of death worldwide. Treating TB is challenging; successful treatment requires ...a long course of multiple antibiotics. Rifampicin (RIF) is a first-line drug for treating TB, and the development of RIF-resistant
M. tuberculosis
makes treatment even more difficult. To determine the mechanism of RIF resistance in these strains, we searched for novel mutations by sequencing. Four isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and unique mutations encoding RpoB G
158
R, RpoB V
168
A, RpoB S
188
P, and RpoB Q
432
insQ, respectively. To evaluate their correlation with RIF resistance, plasmids carrying
rpoB
genes encoding these mutant proteins were transfected into the H
37
Rv reference strain. The plasmid complementation of RpoB indicated that G
158
R, V
168
A, and S
188
P did not affect the MIC of RIF. However, the MIC of RIF was increased in H
37
Rv carrying RpoB Q
432
insQ. To confirm the correlation between RIF resistance and Q
432
insQ, we cloned an
rpoB
fragment carrying the insertion (encoding RpoB Q
432
insQ) into H
37
Rv by homologous recombination using a suicide vector. All replacement mutants expressing RpoB Q
432
insQ were resistant to RIF (MIC > 1 mg/L). These results indicate that RpoB Q
432
insQ causes RIF resistance in
M. tuberculosis
.
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