Biology subjects

Kalia, A.

Publications and source records attributed to Kalia, A..

8 recordsLinked to original sources

bioRxiv2025

Reproducibility and Accuracy of Nanopore-Based Methylome Profiling of Streptococcus dysgalactiae subspecies equisimilis Strains from Cancer Patients

BackgroundDNA methylation influences bacterial gene regulation, virulence, and restriction-modification (RM) systems. Advances by Oxford Nanopore Technologies (ONT) now enable direct methylome profiling from nanopore sequencing using the Dorado basecaller. However, the comparative performance of ONT-only versus hybrid-assembly reference-based methylation calling, particularly regarding genomic DNA quality and inter-operator variability, remains understudied. MethodsSix operators independently prepared fifteen sequencing libraries each for nanopore (MinION R10.4.1 flow cells, Mk1D) and Illumina MiniSeq platforms for two Streptococcus dysgalactiae subsp. equisimilis strains (UT9728, 12 replicates; UT10237, 3 replicates). MicrobeMod v1.0.3 was used to identify methylation and motif profiles using Illumina-corrected hybrid reference assemblies (HRAs) and ONT-only reference assemblies (ORAs). Reproducibility and accuracy were compared using a custom genome annotation feature-enabled modular analysis that mapped and counted methylation site calls to CDS, rRNA and tRNA coordinates. ResultsStrain UT9728 predominantly exhibited N6-methyladenine (6mA) at GATC motifs, whereas strain UT10237 displayed dual methylation patterns: C5-methylcytosine (5mC) at CCWGG motifs and 6mA at GAGNNNNNTAA motifs. Both strains contained Type I and Type II RM systems; UT10237 uniquely harbored a Type IIG RM system with combined restriction and methylation activities. Motif identification concordance using HRAs and ORAs exceeded 99.9%. Reproducibility for methylation calls was high across independent replicates for both HRA (Pearsons r >0.989) and ORA (Pearsons r >0.993) methylation calls in GATC and CCWGG motifs but lower in the GAGNNNNNTAA motif (Pearsons r (HRA) = 0.80; r (ORA) = 0.78). ORA-based methylation site calls for all motifs showed excellent precision and recall compared to HRA-based calls (F1-score >99.999%). ConclusionOur findings support the accuracy, robustness, and utility of ONT-only data based methylome profiling for bacterial epigenetic characterization. Our analytical framework facilitates detailed evaluations of reproducibility and accuracy. Data Summary and AvailabilityO_LIUnprocessed_data_files: DOI: 10.5281/zenodo.15555625 O_LIRaw paired-end FastQ Files (Illumina). C_LIO_LIRaw FastQ Files (ONT; unmodified basecalls). C_LIO_LIRaw uBAM Files (ONT; 6mA_5MC_modified_basecalls). C_LI C_LIO_LIGenome_assemblies_and_annotation_files: DOI: 10.5281/zenodo.15558488 O_LIGenome_Assemblies reconstucted with ONT reads and polished with Illumina Reads (Hybrid assemblies);(strain_name_ _Hyb.gbk) C_LIO_LIGenome_Assemblies reconstructed with ONT reads and polished with ONT reads (ONT-only assemblies); (strain_name_ _ONT.fasta) C_LIO_LIGenBank flatfiles from Hybrid assemblies; (strain_name_ _Hyb.gbk) C_LIO_LIGenbank flatfiles from ONT-only assemblies. (strain_name_ _ONT.gbk) C_LI C_LIO_LIData_Tables and Python_code for modular analysis: DOI: doi.org/10.5281/zenodo.15579791 O_LI9728_10237_ORA+HRA_all_reps_microbemod_output.zip C_LIO_LIMethylation calls mapped to parsed genbank features & feature count matrix files. - [Formula] 9728_HRA_reproducibility_analysis_tables.zip - [Formula] 9728_ORA_reproducibility_analysis_tables.zip - [Formula] 10237_HRA_5mC_6mA_reproducibility_analysis_tables.zip - [Formula] 10237_ORA_reproducibility_analysis_tables.zip C_LIO_LIPython code for annotation feature-based modular analysis - [Formula] methylation_feature_hash_assert_FILTER_9728.py - [Formula] methylation_feature_hash_NOFILTER_9728.py - [Formula] methylation_feature_hash_motif_10237.py - [Formula] hyb_vs_ont_per_replicate_comparison.py - [Formula] hyb_vs_ont_combined_plot.py C_LI C_LIO_LIGenome Assembly_Code Availibility: https://github.com/TSchababerle/Bacterial-Methylation O_LIBash script for automated Hybrid Genome Assembly C_LIO_LIBash Script for automated ONT-Only Assembly C_LI C_LI Impact StatementThis study provides a focused proof-of-principle demonstrating the robustness and reproducibility of Oxford Nanopore Technologies (ONT) sequencing-based methylome profiling without short-read correction. Using independent replicates prepared by multiple operators, we show that nanopore-only methods yield consistent, accurate methylation profiles concordant with short-read corrected ONT reads. This highlights the potential of ONT-only sequencing as a broadly accessible, reliable approach for rapid bacterial epigenomic characterization across diverse clinical contexts.

genomics↗

bioRxiv2024

Advancing protein display on bacterial spores through an extensive survey of coat components

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 proteins, only [~]12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SP investigated, 26 formed functional fusions with {beta}-glucuronidase (GUS) - a [~]272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed the 1) SP promoter activation dynamics during growth, 2) supported GUS activity on spores, 3) surface availability, 4) protection from thermal and proteolytic degradation, and 5) compatibility of co-expression of many of these fusions. Multi-copy expression and pairwise co-expression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and difficulty in predicting compatibility between different SPs fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA (small spore coat assembly protein A) as an effective spore display carrier. SscA supported enzyme activity at least fourfold higher than any other SP, including the well-established and popular anchor, CotY. We attribute this to the biochemical and genetic features of SscA; its promoter demonstrated early and sustained activation relative to other SPs and its small size ([~]3 KDa) likely minimally interferes with enzyme folding, oligomerization, and activity. Although the specific localization of SscA within the spore coat has not been established, its hydrophobic nature and low surface availability suggests that it assembles internally within the spore coat, which makes it highly stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledgebase to advance the biotechnological utility of B. subtilis spores.

synthetic biology↗

bioRxiv2024

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure

BackgroundIt remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant. MethodsCarbapenem mutation frequency was measured in multiple subclades of extended-spectrum {beta}-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/H30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. ResultsAll thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P-value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing blaCTX-M-15 copy numbers via modular, insertion sequence 26 (IS26) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ConclusionsESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/H30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

microbiology↗

bioRxiv2023

Dynamic Periodicity of Extended-Spectrum Cephalosporin Resistant Bloodstream Escherichia coli Infections Driven by Non-CC131 Strains

Extended-spectrum cephalosporin resistant Escherichia coli (ESC-R-Ec) is an urgent public health threat with sequence type clonal complex 131 (STc131), phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R-Ec infections. To address the paucity of recent ESC-R-Ec molecular epidemiology data in the United States, we used whole genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R-Ec at a tertiary care cancer center in Houston, Texas collected from 2016-2020. During the study timeframe, there were 1154 index E. coli bloodstream infections (BSIs) of which 389 (33.7%) were ESC-R-Ec. Using time series analyses, we identified a temporal dynamic of ESC-R-Ec distinct from ESC-susceptible E. coli (ESC-S-Ec), with cases peaking in the last six months of the calendar year. WGS of 297 ESC-R-Ec strains revealed that while STc131 strains accounted for [~]45% of total BSIs, the proportion of STc131 strains remained stable across the study time frame with infection peaks driven by genetically heterogeneous ESC-R-Ec clonal complexes. BlaCTX-M variants accounted for most {beta}-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R-Ec), and amplification of blaCTX-M genes was widely detected in ESC-R-Ec strains, particularly in carbapenem non-susceptible, recurrent BSI strains. BlaCTX-M-55 was significantly enriched within phylogroup A strains, and we identified blaCTX-M-55 plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R-Ec infections at a large tertiary care cancer center and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens. IMPORTANCEGiven that E. coli is the leading cause of worldwide ESC-R Enterobacterales infections, we sought to assess the current molecular epidemiology of ESC-R-Ec using a WGS analysis of many BSIs over a five-year period. We identified fluctuating temporal dynamics of ESC-R-Ec infections, which has also recently been identified in other geographical regions such as Israel. Our WGS data allowed us to visualize the stable nature of STc131 over the study period and demonstrate a limited, but genetically diverse group of ESC-R-Ec clonal complexes are detected during infection peaks. Additionally, we provide a widespread assessment of {beta}-lactamase gene copy number in ESC-R-Ec infections and delineate mechanisms by which such amplifications are achieved in a diverse array of ESC-R-Ec strains. These data suggest that serious ESC-R-Ec infections are driven by a diverse array of strains in our cohort and impacted by environmental factors suggesting that community-based monitoring could inform novel preventative measures.

microbiology↗

bioRxiv2022

Zika virus spreads through infection of lymph node-resident macrophages

Zika virus (ZIKV) is an arthopod-vectored flavivirus that disseminates from the infection site into peripheral tissues, where it can elicit virus-induced pathology. To move through the body, ZIKV is thought to exploit the mobility of myeloid cells, in particular monocytes and dendritic cells. However, multiple distinct steps during viral spread culminate in peripheral tissue infection, and the timing and mechanisms underlying mobile immune cell shuttling of virus remain unclear. To understand the very early steps in ZIKV dissemination from the skin, we kinetically and spatially mapped ZIKV-infected lymph nodes (LNs), an intermediary stop en route to the blood. Contrary to dogma, migratory immune cells were not required for large quantities of virus to reach the LN or blood. Instead, ZIKV rapidly infected a subset of immobile macrophages in the LN, which shed virus through the lymphatic pathway into the blood. Importantly, infection of LN macrophages alone was sufficient to initiate viremia. Together, our studies indicate that sessile macrophages that live and die in the LN contribute to initial ZIKV spread to the blood. These data build a more complete picture of ZIKV movement through the body and identify an alternate anatomical site for potential antiviral intervention. HighlightsO_LIZIKV infects and replicates in distinct LN macrophage populations C_LIO_LILN macrophage infection results in infectious virus in the blood C_LIO_LIVirus reaches the blood in the absence of DC migration or monocyte infection C_LIO_LINodal macrophage infection does not sustain viremia or induce morbidity C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=74 HEIGHT=200 SRC="FIGDIR/small/508424v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@121bc33org.highwire.dtl.DTLVardef@1caf24borg.highwire.dtl.DTLVardef@e94a08org.highwire.dtl.DTLVardef@1fb6f62_HPS_FORMAT_FIGEXP M_FIG Caption: ZIKV infects lymph node macrophages, which shed infectious virus into the lymph and then blood. C_FIG

microbiology↗

bioRxiv2020

Drosha Regulates Oogenesis and microRNAs Germline Autonomously and Non autonomously in C. elegans

Small non-coding RNAs regulate multiple aspects of development including germ cell development. The microRNA pathway genes Dicer, Drosha and Pasha have been shown to regulate oocyte meiotic maturation in C. elegans. However, Dicer controls oocyte meiotic maturation through endo-siRNAs, rather than microRNAs. A repertoire of Drosha-dependent oocyte-expressed microRNAs were identified which regulate various aspects of oogenesis but not oocyte meiotic maturation. These data lead to the following models: (a) microRNAs function redundantly to regulate oocyte meiotic maturation, (b) Drosha and microRNAs function germline non-autonomously to regulate meiotic maturation. We investigated these models and observed that Drosha regulates oocyte meiotic maturation in a germline non-autonomous manner. Additionally, we uncovered a role for Drosha in regulating pachytene progression and oocyte development in a germline autonomous manner through miR-35 family and miR-51 respectively. Interestingly we also find that though Drosha-dependent oocyte-expressed microRNAs, miR-61 and miR-72, are sufficient to regulate pachytene progression and oocyte development respectively, they are generated in a germline non-autonomous manner. Collectively these data reveal a Drosha-dependent microRNA circuit, which coordinates oocyte development germline autonomously as well as through soma-germline communication.

developmental biology↗

bioRxiv2019

β-lactamase Amplification and Porin Loss Drive Progressive β-lactam Resistance in Recurrent ESBL Enterobacteriaceae Bacteremia

BackgroundCarbapenem resistant Enterobacterales (CRE) remain urgent antimicrobial resistance threats. Approximately half of CRE clinical isolates lack carbapenem hydrolyzing enzymes and develop carbapenem resistance through alternative mechanisms. The purpose of this study was to elucidate the development of carbapenem resistance mechanisms from clonal, recurrent extended-spectrum {beta}-lactamase positive Enterobacterales (ESBL-E) bacteremia isolates in a vulnerable patient population. MethodsThis study investigated a historical, retrospective cohort of ESBL-E bacteremia cases in the University of Texas MD Anderson Cancer Center (MDACC) from January 2015 to July 2016. Phylogenetic and comparative genomic analyses were performed to identify clonal, recurrent ESBL-E isolates developing carbapenem resistance. Oxford Nanopore Technology (ONT) long-read and Illumina short-read sequencing data were used to generate consensus assemblies and to identify signatures of mobile genetic element mediated amplification and transposition of antimicrobial resistance genes. Serial passaging experiments were performed on a set of clinical ST131 ESBL-E isolates to recapitulate in vivo observations. qPCR and qRT-PCR were used to determine respective copy number and transcript levels of {beta}-lactamase genes. Results116 ESBL-E bacteremia cases were identified, 16 of which had documented recurrent infections. Four serial, recurrent isolates displayed a carbapenem resistant phenotype, three without the acquisition of a known carbapenemase. These three isolates had non-carbapenemase-producing CRE (non-CP-CRE) mechanisms driven by IS26- and ISEcp1-mediated amplification of respective translocatable units (TU) and transposition units (TPU) harboring both blaOXA-1 and blaCTX-M variants with concomitant outer membrane porin disruption. The TU and TPU structures inserted into the open reading frames of outer membrane porin genes in a subset of non-CP-CRE isolates. Serial passage of an index ST131 ESBL-E isolate under selective carbapenem exposure resulted in chromosomal amplification of modular, TUs harboring {beta}-lactamase genes with concomitant porin inactivation, recapitulating the in vivo carbapenem resistance progression. Long-read sequencing of two additional MDACC bacteremia strains identified similar non-CP-CRE mechanisms observed in the serial isolates. ConclusionsNon-CP-CRE de novo mechanisms were the primary driver of CRE development in recurrent bacteremia cases within this vulnerable patient population. The incorporation of long-read ONT data into AMR surveillance platforms is critical to identify high-risk CRE isolates that are difficult to identify with low-resolution phenotypic and molecular characterization methods.

genomics↗

bioRxiv2018

Identification of a Chimeric M4 Protein and Novel Emm Pattern in Currently Circulating Strains of Emm4 Group A Streptococcus

Group A Streptococcus (GAS) is classified by sequence of the gene encoding the M protein (emm) and the patterns into which emm types are grouped. We discovered a novel emm pattern in emm4 GAS, historically considered pattern E, arising from a fusion event between emm and the adjacent enn gene. We identified the emm/enn fusion event in 51/52 emm4 GAS strains isolated by national surveillance in 2015. GAS isolates with an emm/enn fusion event completely replaced pattern E emm4 strains over a 4-year span in Houston (2013-2017). The novel emm/enn gene fusion and new emm pattern has potential vaccine implications.

genomics↗