Biology subjects

Salama, N.

Publications and source records attributed to Salama, N..

7 recordsLinked to original sources

bioRxiv2026

CagA delivery by the Helicobacter pylori Cag-Type IV Secretion System confers fitness benefits and costs during stomach infection

Helicobacter pylori strains encoding the cag-pathogenicity island (cag-PAI) and the effector toxin cagA are associated with worse disease outcomes. The cag-PAI encodes the Cag type IV secretion system (Cag-T4SS) which injects CagA and other bacterial products into gastric epithelial cells. Prior work revealed that host adaptive immunity promotes recombination in the cag-PAI gene cagY to attenuate Cag-T4SS activity during chronic infection, suggesting a fitness cost to assembling an active Cag-T4SS. To explore potential selective benefits and costs for the Cag-T4SS and CagA, we employed single strain and competitive infections at both acute and chronic timepoints in wildtype mice and transgenic mice that either attenuate innate immune responses or promote gastric pathology independent of H. pylori infection to examine the relative fitness of mutant H. pylori strains. Our results suggest that an active Cag-T4SS and CagA confer a fitness benefit during initial colonization through Cag-T4SS activity-dependent epithelial cell interactions that activate cancer-related signaling pathways. However, increasing gastric inflammation confers a fitness cost to CagA translocation, promoting Cag-T4SS shutoff. Targeted and whole genome sequencing revealed multiple mechanisms of Cag-T4SS attenuation, with recombination-mediated changes in cagY prevalent at early timepoints and mutations in a variety of Cag-T4SS structural genes accumulating as disease progresses. The need for Cag-T4SS activity and CagA translocation during initial gland colonization likely underlies the mutational pattern observed. Collectively this work reveals new insights into selective constraints on the H. pylori Cag-T4SS as well as resultant genetic adaptation processes that lead to retention of the cag-PAI and virulence.

microbiology↗

bioRxiv2026

CD95/Fas Apoptosis Signal Initiation Depends on the Ligand Oligomerization State and Formation of Small Ligand-Receptor Complexes

The death receptor cluster of differentiation 95 (CD95 / Fas) is an important inducer of apoptotic activity in tumor cells, but may exhibit differential cell responses (ranging from predominantly apoptosis up to proliferation) when stimulated by its CD95 ligand (CD95L/FasL). How ligand form and receptor-ligand stoichiometry shape these divergent outcomes remain unresolved. To define structural and functional determinants of CD95 signaling, we systematically compared the oligomerization and activity of native CD95L with CD95L variants, where ligand trimers were stabilized either by a FLAG-tag for subsequent monoclonal antibody (mAb) crosslinking or by genetically fused isoleucine zippers (IZ). Apoptotic activity varied markedly with both ligand concentration and ligand variant type. To quantify receptor-ligand stoichiometry at the cell membrane, we combined quantitative Stimulated Emission Depletion (qSTED) nanoscopy, simulations, and biochemical assays. Across conditions, signaling complexes consisted of small oligomeric assemblies, with up to three CD95 receptors engaging a trimeric ligand. Despite similar stoichiometry, biochemical measurements revealed substantial differences in ligand oligomerization and binding affinity, with IZ-CD95L exhibiting markedly higher affinity than FLAG-CD95L (KD of 0.81 nM and 18.4 nM, respectively). Dimeric/trimeric CD95 formed by CD95L complexation or bridged CD95 by mAb enabled flexible intracellular FADD linkage for enhanced signaling.These results indicate that apoptotic potency is governed by the proximity of CD95 receptors, achieved through receptor bridging as well as by a stabilized trimeric ligand, both of which enhance receptor recruitment and binding avidity. Our findings provide mechanistic insight into CD95 signaling and suggest strategies for optimized apoptosis-inducing therapeutics. Significance StatementThe CD95/Fas receptor-ligand system is a central regulator of programmed cell death, yet the earliest molecular events of CD95 activation remain unclear. We systematically compared different CD95L/FasL variants and show that ligand architecture critically shapes receptor engagement and death signaling. An engineered FasL with an isoleucine zipper exhibited larger trimeric lig- and fractions, stability, and receptor binding, resulting in stronger apoptotic activity. Super-resolution imaging and simulations indicate that CD95 is mainly monomeric at the membrane and forms small oligomers after ligand binding. The flexibility of intracellular FADD linkage trigerred by dimeric/trimeric or bridged CD95 enables more intense signaling. These findings clarify how ligand structure governs CD95 activation and provide design principles for death receptor-targeted therapeutics.

biophysics↗

bioRxiv2025

Metaplasia Enables Stomach Colonization by Fusobacterium animalis

Infection with Helicobacter pylori is the major risk factor for gastric cancer worldwide; yet the exact mechanisms behind this link remain unclear. H. pylori-associated tissue changes often disrupt the gastric microbiome, enabling secondary gastric colonization by oral bacteria. Among these secondary colonizers, Fusobacterium species have documented associations with several gastrointestinal cancers. We found that both F. animalis and F. nucleatum invade cultured human gastric adenocarcinoma cells, but F. animalis exhibited higher adherence and invasion, and hypoxic conditions promoted higher bacterial survival. Both adherence and invasion were inhibited by exogenous GalNAc, a glycan commonly observed in membrane glycoproteins of adenocarcinoma cells, and a target of the fusobacterial adhesin Fap2. Using a mouse model of gastric metaplasia, we found that F. animalis colonized gastric tissue only after metaplasia onset, growing in multispecies biofilms in the mucus layer, while F. nucleatum colonized neither healthy nor metaplastic gastric tissue. Metaplasia led to upregulation of Gal-GalNAc in the stomach, and reduced gastric acidity allowed higher F. animalis loads in this model. By contrast, inflammation and the presence of H. pylori did not significantly influence stomach colonization by F. animalis. Overall, our data support a model in which H. pylori-induced metaplasia makes the stomach susceptible to secondary infection by another cancer-associated microbe, F. animalis.

microbiology↗

bioRxiv2025

Muc6-expressing gastric isthmus progenitors contribute to regeneration and metaplasia supported by myeloid-mesenchymal interactions

Gastric mucosal homeostasis is maintained by tissue-resident stem and progenitor cells residing in the isthmus region. Following mucosal injury, surviving cells contribute to regeneration, coinciding with characteristic pathological changes such as atrophic gastritis and metaplasia. To comprehensively understand the cellular dynamics involved in this process, we performed single-cell and spatial transcriptomics using newly generated transgenic mice. In human samples and mouse models, loss of gastric chief cells precedes, and even induces, loss of parietal cells during the progression of atrophy and metaplasia, validating the causal relationship underlying the decrease of these two lineages. Single-cell analysis confirmed robust stemness and metaplastic changes in the Muc6-expressing neck lineage following either chief or parietal cell ablation, and lineage-tracing experiments revealed that Muc6-expressing isthmus progenitors serve as a source of metaplasia and regeneration. Mechanistically, mucosal injury recruits IL-1-expressing myeloid cells, which stimulates NRG1 production in stromal fibroblasts, leading to mucosal proliferation and regeneration mediated by Myc activation in isthmus progenitors. These findings highlight the injury-responsible stem cell-like function of Muc6-expressing isthmal progenitors, which play a critical role in mucosal homeostasis and disease progression. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/648856v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@10fc2eaorg.highwire.dtl.DTLVardef@1c5b82corg.highwire.dtl.DTLVardef@1be747dorg.highwire.dtl.DTLVardef@d1d722_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Distinct regions of H. pylori's bactofilin regulate protein interactions to control cell shape

The helical shape of H. pylori cells promotes robust stomach colonization, however, how the helical shape of H. pylori cells is determined is unresolved. Previous work identified helical-cell-shape-promoting protein complexes containing a peptidoglycan-hydrolase (Csd1), a peptidoglycan precursor synthesis enzyme (MurF), a non-enzymatic homologue of Csd1 (Csd2), non-enzymatic transmembrane proteins (Csd5 and Csd7), and a bactofilin (CcmA). Bactofilins are highly conserved, spontaneously polymerizing cytoskeletal bacterial proteins. We sought to understand CcmAs function in generating the helical shape of H. pylori cells. Using CcmA deletion analysis, in vitro polymerization, and in vivo co-immunoprecipitation experiments we identified that the bactofilin domain and N-terminal region of CcmA are required for helical cell shape and the bactofilin domain of CcmA is sufficient for polymerization and interactions with Csd5 and Csd7. We also found that CcmAs N-terminal region inhibits interaction with Csd7. Deleting the N-terminal region of CcmA increases CcmA-Csd7 interactions and destabilizes the peptidoglycan-hydrolase Csd1. Using super-resolution microscopy, we found that Csd5 recruits CcmA to the cell envelope and promotes CcmA enrichment at the major helical axis. Thus, CcmA helps organize cell-shape-determining proteins and peptidoglycan synthesis machinery to coordinate cell wall modification and synthesis, promoting the curvature required to build a helical cell.

microbiology↗

bioRxiv2021

Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the CNS, were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, viral genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

immunology↗

bioRxiv2020

Helicobacter pylori accelerates KRAS-dependent gastric dysplasia

More than 80% of gastric cancer is attributable to stomach infection with Helicobacter pylori (Hp), even though the bacterium is not always present at time of diagnosis. Infection is thought to lead to cancer by promoting the accumulation of oncogenic mutations downstream of inflammation; once oncogenic pathways become activated, infection may become dispensable for cancer development. Gastric preneoplastic progression involves sequential changes to the tissue, including loss of parietal cells, spasmolytic polypeptide-expressing metaplasia (SPEM), intestinal metaplasia (IM) and dysplasia. In mice, active KRAS expression recapitulates these tissue changes in the absence of Hp infection. This model provides an experimental system to investigate whether Hp infection has additional roles in preneoplastic progression, beyond initiating inflammation. Mice were assessed by evaluating tissue histology, gene expression changes, the immune cell repertoire, and expression of metaplasia and dysplasia markers. Compared to Hp-/KRAS+ mice, Hp+/KRAS+ mice had i) severe T cell infiltration and altered macrophage polarization; ii) altered expression of metaplasia markers, including increased expression of CD44v9 (SPEM) and decreased expression of TFF3 (IM); iii) more dysplastic (TROP2+) glands; and iv) greater proliferation of metaplastic and dysplastic glands. Hp was able to persistently colonize the stomach during the onset of these tissue changes, and eradication of Hp with antibiotics prevented metaplastic, dysplastic and proliferation marker changes. Collectively, these results suggest that gastric preneoplastic progression differs between Hp+ and Hp-cases, and that sustained Hp infection can promote the later stages of gastric preneoplastic progression, in addition to its established role in initiating chronic inflammation.

cancer biology↗