What is S. Typhi HlyE and how was it discovered?

S. Typhi HlyE is a hemolysin protein encoded by the hlyE gene located within Salmonella Pathogenicity Island 18 (SPI-18), a small 2.3kb genomic island present in S. Typhi but absent in S. Typhimurium . This protein belongs to the hemolysin family and is related to the Escherichia coli K12 HlyE hemolysin . The discovery of HlyE emerged from comparative genomic analyses between S. Typhi and S. Typhimurium, which revealed the presence of SPI-18 in S. Typhi and its absence in S. Typhimurium . PCR assays subsequently confirmed that SPI-18 is present in S. Typhi and in many, but not all, serovars of S. enterica subsp. enterica from the SARB collection .

What is the genetic organization of SPI-18 and the hlyE gene?

SPI-18 is a 2.3kb genomic island that contains two potential genes, with hlyE being one of them . The hlyE gene encodes a hemolysin protein related to E. coli K12 HlyE hemolysin . Interestingly, almost all serovars of the SARB collection that cause systemic infections in humans possess SPI-18 and the hlyE gene, and they express an active hemolysin that can be revealed when the bacterial envelope is destabilized . This suggests that the genetic organization of SPI-18 and the presence of hlyE may be associated with the ability of certain Salmonella serovars to cause systemic disease in humans.

How does S. Typhi HlyE activity manifest in laboratory conditions?

• Upon lysis of bacterial cells with bacteriophages

• In the presence of ampicillin

• In an ompA genetic background

These conditions suggest that HlyE activity is normally suppressed or contained by the bacterial envelope structure in S. Typhi . This unusual property of requiring envelope destabilization for activity detection presents unique challenges for researchers studying this virulence factor.

What are the recommended methods for detecting HlyE activity in S. Typhi?

Standard plate assays are insufficient for detecting S. Typhi HlyE activity in laboratory conditions . Instead, researchers should employ techniques that compromise the bacterial envelope integrity:

When testing for HlyE activity, researchers should incorporate appropriate controls, including known HlyE-positive and HlyE-negative strains, to validate their experimental setup .

How can researchers effectively study the role of HlyE in S. Typhi invasion of epithelial cells?

To investigate HlyE's role in epithelial cell invasion, researchers should employ a multifaceted approach:

• Genetic manipulation: Generate precise hlyE deletion mutants in S. Typhi using techniques such as lambda Red recombination system or CRISPR/Cas9-based approaches.

• Cell culture models: Human epithelial cell lines (HeLa, Caco-2, HT-29) serve as appropriate in vitro models. Invasion assays should be performed by infecting epithelial cell monolayers with wild-type S. Typhi and isogenic hlyE mutants at various multiplicities of infection (MOI).

• Quantification methods: Use gentamicin protection assays to eliminate extracellular bacteria and count intracellular bacteria through serial dilution and plating, or by fluorescence microscopy using tagged strains.

• Complementation studies: To confirm phenotypes are specifically due to hlyE loss, complement the mutation by reintroducing the wild-type hlyE gene on a plasmid or by chromosomal integration .

S. Typhi hlyE mutants have been demonstrated to be impaired in invasion of human epithelial cells in vitro, confirming this gene's role in the invasion process .

What approaches can be used to study HlyE expression regulation in S. Typhi?

Understanding HlyE expression regulation requires multiple experimental approaches:

• Transcriptional analysis: Implement quantitative RT-PCR to measure hlyE mRNA levels under various conditions (pH changes, oxygen limitation, nutrient deprivation, host cell contact). RNA-seq can provide comprehensive transcriptome analysis during infection of human macrophages and epithelial cells .

• Promoter analysis: Create reporter gene fusions (e.g., hlyE promoter-lacZ or -gfp) to identify environmental and genetic factors influencing expression. Chromatin immunoprecipitation (ChIP) assays can identify transcription factors binding to the hlyE promoter.

• Protein detection: Develop specific antibodies against HlyE or epitope-tag the protein for detection by Western blotting, immunofluorescence, or ELISA.

• Host-pathogen interface studies: Employ infection models using human-derived enteroid systems to study transcriptional responses during infection. This approach has revealed that S. Typhi can downregulate critical host genes responsible for bacterial clearance, host immunity, and cytoskeleton rearrangements .

What evidence supports HlyE as a virulence determinant in S. Typhi?

Multiple lines of evidence establish HlyE as a significant virulence determinant in S. Typhi:

• Invasion defects: S. Typhi hlyE mutants show impaired invasion of human epithelial cells in vitro, demonstrating its role in the initial stage of infection .

• Heterologous expression effects: When the S. Typhi hlyE gene is transferred to S. Typhimurium (which naturally lacks this gene), the recipient strain shows improved colonization of deep organs in mouse models, indicating HlyE's contribution to systemic spread .

• Phylogenetic distribution: Almost all Salmonella serovars from the SARB collection that cause systemic infections in humans possess SPI-18 and an active hlyE gene, suggesting evolutionary selection for this virulence factor in serovars that cause invasive disease .

• Diagnostic potential: The recognition of HlyE as a specific antigen for detecting S. Typhi infections further supports its expression during human infection and its potential role in pathogenesis .

These findings collectively confirm that HlyE functions as a virulence determinant that contributes to S. Typhi's ability to invade host cells and establish systemic infection.

How does S. Typhi HlyE interact with host cell components during infection?

The interaction between S. Typhi HlyE and host cell components involves several mechanisms:

Further research using advanced techniques like proximity labeling, co-immunoprecipitation, and high-resolution imaging is needed to fully characterize HlyE's molecular interactions with host components.

How do immune responses to S. Typhi HlyE differ between human and mouse models?

Understanding immune responses to S. Typhi HlyE in different host species is complicated by S. Typhi's host restriction to humans:

• Human immune response: In human hosts, S. Typhi infection can activate inflammasomes in monocyte-derived macrophages within two hours post-infection in a SPI-1-dependent manner . HlyE likely contributes to this process, as it is expressed and immunogenic during human infection, evidenced by the development of specific antibodies that can be detected in typhoid patients .

• Mouse models limitations: Standard mouse models are not naturally susceptible to S. Typhi infection due to host restriction mechanisms. This restriction can be attributed to:

  • Absence of specific virulence genes like gtgE in S. Typhi

  • Pseudogenization of SopD2 in S. Typhi

  • The action of the Rab32-BLOC3 pathway in murine macrophages and dendritic cells, which delivers antimicrobial compounds like itaconate into Salmonella-containing vacuoles

• Heterologous expression studies: When S. Typhi HlyE is expressed in S. Typhimurium, it enhances colonization of deep organs in mice, suggesting that HlyE can function across species barriers and potentially modulate immune responses .

• Humanized mouse models: Recent advances in developing humanized mouse models may provide better platforms for studying specific immune responses to S. Typhi HlyE, though these models still have limitations in fully recapitulating human immune system complexity .

How can DNA aptamers targeting HlyE improve typhoid fever diagnostics?

DNA aptamers targeting S. Typhi HlyE represent a promising approach for improving typhoid fever diagnostics, offering several advantages over traditional methods:

• High specificity and affinity: Recently developed aptamers against S. Typhi HlyE have demonstrated excellent specificity, clearly distinguishing S. Typhi HlyE from other bacteria including Salmonella Paratyphi A, Salmonella Paratyphi B, Shigella flexneri, Klebsiella pneumonia, and Escherichia coli . The highest binding affinity was observed for AptHlyE97 with a Kd value of 83.6 nM .

• Advantages over antibodies: Aptamers offer numerous benefits compared to antibodies, including:

  • Quicker generation time

  • Reduced manufacturing costs

  • Minimal batch-to-batch variability

  • Greater modifiability

  • Improved thermal stability

• Potential for point-of-care tests: These aptamers can be utilized as diagnostic ligands for the development of inexpensive and effective point-of-care tests, which are particularly valuable in developing countries where typhoid is endemic .

• Overcoming current diagnostic limitations: Conventional diagnostic methods like the Widal test have significant drawbacks, including cross-reactivity with different serovars, low sensitivity, and cross-reactivity with unrelated diseases like malaria, dengue fever, and brucellosis . Aptamer-based diagnostics could potentially overcome these limitations.

What methodologies are used to develop and characterize aptamers against S. Typhi HlyE?

The development and characterization of aptamers against S. Typhi HlyE involve several sophisticated methodologies:

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX):

  • Initial selection from a large random oligonucleotide library

  • Iterative rounds of binding, partitioning, and amplification

  • Enrichment for sequences with high affinity and specificity for HlyE

• Enzyme-Linked Oligonucleotide Assay (ELONA):

  • Assessment of binding affinity and specificity

  • Quantitative measurement of aptamer-target interactions

  • Determination of dissociation constants (Kd values)

• Selectivity Testing:

  • Cross-reactivity assessment against related bacterial antigens

  • Validation using antigens from Salmonella Paratyphi A, Salmonella Paratyphi B, Shigella flexneri, Klebsiella pneumonia, and Escherichia coli

• Binding Kinetics Analysis:

  • Determination of association and dissociation rates

  • Calculation of equilibrium dissociation constants

  • Establishment of nanomolar binding affinities

These methodologies have successfully identified aptamers like AptHlyE11, AptHlyE45, and AptHlyE97 with excellent specificity and affinity for S. Typhi HlyE, making them promising candidates for diagnostic application .

How do HlyE-based diagnostics compare with current serological methods for typhoid fever detection?

HlyE-based diagnostics offer several advantages over current serological methods for typhoid fever detection:

The current limitations of serological methods like the Widal test include:

• Cross-reactivity with different Salmonella serovars

• Low sensitivity and variable specificity

• False positives with unrelated diseases like malaria, dengue fever, and brucellosis in endemic areas

HlyE-based diagnostics, particularly those using aptamers, have the potential to overcome these limitations by:

• Providing higher specificity (demonstrated against multiple bacterial species)

• Offering more stable diagnostic reagents with lower batch-to-batch variability

• Being potentially adaptable to point-of-care formats suitable for resource-limited settings

How does host genetic variation affect susceptibility to S. Typhi HlyE-mediated pathogenesis?

Host genetic factors significantly impact susceptibility to S. Typhi infection, with potential implications for HlyE-mediated pathogenesis:

• MCOLN2 variation: Recent cellular genome-wide association studies have identified human genetic variation affecting the divalent cation channel mucolipin-2 (MCOLN2 or TRPML2) that influences S. Typhi intracellular replication. This channel restricts bacterial growth through magnesium deprivation, conducting Mg²⁺ currents out of endolysosomes . Genetic polymorphisms that alter MCOLN2 function may directly impact host resistance to S. Typhi, potentially modifying responses to HlyE.

• Innate immune response variation: Genetic differences in pathogen recognition receptors, inflammasome components, or downstream signaling pathways may alter the host response to HlyE. S. Typhi can activate the inflammasome in monocyte-derived macrophages two hours post-infection in a SPI-1-dependent manner , suggesting that genetic variation in this pathway could influence disease outcomes.

• Adaptive immune response genetics: HLA haplotypes and other immunogenetic factors likely influence the quality and magnitude of adaptive immune responses to S. Typhi antigens, including HlyE. This variation could affect both the acute response to infection and long-term protective immunity.

• Rab32–BLOC3 pathway: In murine macrophages and dendritic cells, the Rab32–BLOC3 pathway delivers itaconate, a mitochondria-derived antimicrobial metabolite, into Salmonella-containing vacuoles to restrict S. Typhi growth . Human genetic variation in this pathway could potentially modify susceptibility to S. Typhi infection.

Future research should focus on identifying specific human genetic variants that modify responses to HlyE and determining whether these variants correlate with clinical outcomes in typhoid fever.

What are the structural and functional differences between S. Typhi HlyE and homologous proteins in other enteric pathogens?

S. Typhi HlyE shares homology with hemolysins in other enteric bacteria, but has distinctive characteristics:

• Structural comparisons:

  • S. Typhi HlyE is related to E. coli K12 HlyE hemolysin

  • Detailed structural analysis of S. Typhi HlyE is needed to identify unique features that may contribute to its role in typhoid pathogenesis

  • Protein modeling and crystallography studies could reveal conformational differences that affect activity or host interactions

• Functional distinctions:

  • Unlike many other bacterial hemolysins, S. Typhi HlyE activity cannot be detected by standard plate assays and only becomes evident under conditions that compromise the bacterial envelope

  • Heterologous expression of S. Typhi HlyE in S. Typhimurium improves colonization of deep organs in mice, suggesting functional capabilities beyond typical hemolytic activity

  • The precise mechanism by which HlyE contributes to epithelial cell invasion requires further characterization

• Evolutionary considerations:

  • SPI-18 containing hlyE is present in S. Typhi and many, but not all, serovars of S. enterica subsp. enterica from the SARB collection

  • Almost all serovars that cause systemic infections in humans possess SPI-18 and hlyE

  • The selective pressure maintaining functional hlyE in typhoidal Salmonella suggests important roles in host-specific pathogenesis

Comparative analysis between S. Typhi HlyE and homologous proteins using techniques like site-directed mutagenesis, chimeric protein construction, and heterologous expression systems would help identify critical functional domains and species-specific adaptations.

How can advanced in vitro and in vivo models be leveraged to study S. Typhi HlyE in host-pathogen interactions?

Several advanced models can be used to study S. Typhi HlyE in complex host-pathogen interactions:

• Human Organoid Models:

  • Adult stem cells (ASCs) and pluripotent stem cells (PSCs) derived human-enteroid models provide physiologically relevant intestinal environments

  • Human-derived enteroid model systems using human tissue biopsies and human ileum allow transcriptional profiling of both bacteria and host tissue

  • These systems have revealed that S. Typhi downregulates critical host genes responsible for bacterial clearance, host immunity, and cytoskeleton rearrangements

• Humanized Mouse Models:

  • Transgenic expression of human receptors or immune components in mice

  • Transposon-directed insertion site sequencing (TraDIS) in humanized mouse models has provided insights into S. Typhi virulence factors

  • These models have shown that SPI-2-encoded T3SS of S. Typhi is dispensable for the induction of systemic typhoid fever

• Advanced Cell Culture Systems:

  • Human monocyte-derived macrophages and THP-1 cells for studying intracellular survival

  • Co-culture systems that incorporate multiple cell types to mimic tissue environments

  • Studies have shown that type IVB pili encoded by the pil operon in SPI-7 of S. Typhi can augment the expression of NF-κB and proinflammatory cytokine IL-6 in THP-1 cells

• Alternative Model Organisms:

  • Caenorhabditis elegans has emerged as a model system for S. Typhi research

  • While not mimicking human disease perfectly, these models can provide insights into conserved host-pathogen interactions

• High-resolution Imaging Techniques:

  • Live-cell imaging to track HlyE localization during infection

  • Super-resolution microscopy to visualize interactions with host cell structures

  • Correlative light and electron microscopy to link protein localization with ultrastructural changes

These advanced models provide powerful tools for dissecting the complex roles of HlyE in S. Typhi pathogenesis beyond what can be observed in traditional cell culture or animal models.