Metabolic dysfunction-associated steatotic liver disease related cirrhosis and incidence of portal vein thrombosis.

BACKGROUND: There is heterogeneous data on whether metabolic-associated steatohepatitis is an independent risk factor for portal vein thrombosis (PVT). We aim to compare the incidence of PVT in patients with cirrhosis with and without metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: This is a single-center retrospective study of patients with cirrhosis seen between 1 January 2016 and 31 January 2021. Patients with a history of hepatocellular cancer, liver transplant, Budd-Chiari syndrome, and intra-abdominal malignancies were excluded. Patients with cirrhosis were followed from their first hepatology visit for 180 days to determine the incidence of PVT. Cox proportional hazard regression was used to determine the relationship between MASLD with PVT. RESULTS: We analyzed data from 2785 patients with cirrhosis who met inclusion and exclusion criteria [mean age: 61.0 ±â€…12.3 years, 44.3% female, 63.8% Whites and mean model for end-stage liver disease-sodium (MELD-Na) score: 11.7 ±â€…6.1]. MASLD was present in 21.7% of patients. A total of 89 patients developed PVT during the follow-up, which was fewer in patients with MASLD [2.0% vs. 3.5%, P = 0.04, unadjusted heart rate (HR): 0.60, 95% confidence interval (CI): 0.27-0.96, P = 0.04]. After adjusting for the demographics, MASLD-related comorbid conditions and MELD-Na score, MASLD was associated with a lower incidence of PVT as compared to non-MASLD cirrhosis (HR: 0.44, 95% CI: 0.21-0.92, P = 0.03). After adjusting for the indicators of Child-Pugh Turcotte score, the risk of PVT in patients with MASLD compared to non-MASLD was not statistically significant (HR: 0.50, 95% CI: 0.22-1.13, P = 0.096). CONCLUSION: PVT incidence was lower in patients with MASLD cirrhosis as compared to non-MASLD cirrhosis. However, the difference was not significantly different after adjusting for liver decompensation.

Cross-domain information fusion for enhanced cell population delineation in single-cell spatial-omics data.

Cell population delineation and identification is an essential step in single-cell and spatial-omics studies. Spatial-omics technologies can simultaneously measure information from three complementary domains related to this task: expression levels of a panel of molecular biomarkers at single-cell resolution, relative positions of cells, and images of tissue sections, but existing computational methods for performing this task on single-cell spatial-omics datasets often relinquish information from one or more domains. The additional reliance on the availability of "atlas" training or reference datasets limits cell type discovery to well-defined but limited cell population labels, thus posing major challenges for using these methods in practice. Successful integration of all three domains presents an opportunity for uncovering cell populations that are functionally stratified by their spatial contexts at cellular and tissue levels: the key motivation for employing spatial-omics technologies in the first place. In this work, we introduce Cell Spatio- and Neighborhood-informed Annotation and Patterning (CellSNAP), a self-supervised computational method that learns a representation vector for each cell in tissue samples measured by spatial-omics technologies at the single-cell or finer resolution. The learned representation vector fuses information about the corresponding cell across all three aforementioned domains. By applying CellSNAP to datasets spanning both spatial proteomic and spatial transcriptomic modalities, and across different tissue types and disease settings, we show that CellSNAP markedly enhances de novo discovery of biologically relevant cell populations at fine granularity, beyond current approaches, by fully integrating cells' molecular profiles with cellular neighborhood and tissue image information.

GlycA: Evaluation of a New Biomarker of Acute Pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a leading cause of gastrointestinal hospital admissions, with up to 40% mortality in patients with moderate-severe AP. Glycoprotein acetylation (GlycA) is measured as a nuclear magnetic resonance signal (NMR) of the post-translational modification of glycosylated acute-phase proteins released during inflammation. We aimed to investigate the role of GlycA as an inflammatory biomarker of AP. METHODS: We prospectively enrolled 20 AP patients and 22 healthy controls and collected EDTA plasma samples at admission and discharge. NMR spectra were acquired from these samples using a 400 MHz Vantera® Clinical Analyzer, and GlycA concentrations were calculated (normal = 400 µmol/L). The GlycA NMR signal, at 2.00 ± 0.01 ppm in the NMR spectrum, is derived from the N-acetyl methyl group protons within the carbohydrate side chains of circulating glycoproteins such as α1-acid glycoprotein, haptoglobin, α1-antitrypsin, α1-antichymotrypsin, and transferrin. GlycA levels were then compared between AP patients and controls, as well as within the AP group, based on etiology and severity. RESULTS: Demographic comparisons were similar, except for a higher BMI in AP patients compared to healthy controls (29.9 vs. 24.8 kg/m2; p < 0.001). AP was mild in 10 patients, moderate in 7, and severe in 3. GlycA levels were higher in AP patients than healthy controls on admission (578 vs. 376 µmol/L, p < 0.001) and at discharge (655 vs. 376 µmol/L, p < 0.001). GlycA levels were significantly higher in patients with moderate-severe AP than in those with mild AP at discharge (533 vs. 757 µmol/L, p = 0.023) but not at admission. After adjusting for BMI, multivariable regression indicated that patients with GlycA levels > 400 µmol/L had significantly higher odds of having AP of any severity (OR = 6.88; 95% CI, 2.07-32.2; p = 0.004) and mild AP (OR = 6.12; 95% CI, 1.48-42.0; p = 0.025) than controls. CONCLUSION: Our pilot study highlights the use of GlycA as a novel diagnostic biomarker of inflammation in patients with AP. Our study shows that GlycA levels were significantly higher in hospitalized AP patients compared to healthy controls. Patients with moderate-to-severe AP had higher GlycA levels compared to patients with mild AP at the time of their hospital discharge, suggesting persistent inflammation in patients with severe disease.

Associations between alcohol consumption and hepatic steatosis in the USA.

Despite being widely recognized as a common cause of fatty liver, the exact impact of alcohol consumption on hepatic steatosis in the general population is elusive. The recent National Health and Nutrition Examination Survey (NHANES) allowed us to examine this relationship among US adults. Herein, we extracted data on detailed alcohol consumption and controlled attenuation parameter (CAP) by FibroScan from 4509 participants in NHANES 2017-2018. Compared to metabolic risk factors such as diabetes and obesity, the association between alcohol consumption and CAP was less significant. In multivariable analysis, only those drinking 5-7 times per week showed significant increases in CAP scores. Although both frequency and quantity of drinking were positively associated with CAP score, only frequency remained significant after adjustment for quantity and binge drinking. These epidemiological observations suggested that the impact of alcohol on hepatic steatosis was much smaller than metabolic factors and dependent upon the frequency of drinking.

Further Defining the 2012 Multi-Society Task Force Guidelines for Surveillance of High-risk Adenomas: Is a 3-Year Interval Needed for All Patients?

GOALS: We set out to determine whether variation from this 3-year follow-up interval was associated with the finding of subsequent high-risk adenoma (HRA). BACKGROUND: HRAs include the following: (1) an adenoma measuring ≥10 mm, (2) ≥3 adenomas found during a single procedure, and (3) an adenoma with high-grade dysplasia or villous architecture. The current Multi-Society Task Force guideline for timing of surveillance colonoscopy after removal of a HRA is 3 years. STUDY: In 2016, we analyzed 495 patients who had a HRA removed during a 2008 colonoscopy. We compared the frequency of finding another HRA at follow-up intervals. We used the current guidelines as our referent group and performed logistical regression to identify whether any patient characteristics, procedural factors, or type of HRA predicted the development of HRAs on follow-up colonoscopy. RESULTS: Individuals who followed-up at a median of 4.5 years did not have more HRA on follow-up compared with those who followed-up at 3 years (25.2% vs. 21.0%, P=0.062). These groups had similar baseline characteristics. Older individuals, male gender, having a history of polyps, and piecemeal resection of an HRA predicted future HRAs. The removal of ≥3 adenomas in 2008 as well as a combination of multiple, large, and advanced polyps showed a higher risk of future HRAs. CONCLUSIONS: The 2012 Multi-Society Task Force recommendation of 3-year follow-up after removal of HRAs may not apply to all patients. We showed that a combination of patient demographics, procedural factors, and pathology best determines the surveillance colonoscopy interval.

Posttransplant Lymphoproliferative Disorder Isolated to the Adrenal Gland in a Liver Transplant Patient.

Posttransplant lymphoproliferative disorder (PTLD) is a serious complication that accounts for up to 20% of malignancies after solid organ transplantation. We describe a rare case of isolated PTLD in the adrenal gland occurring 7 months after liver transplant in a patient who developed a primary Epstein-Barr virus infection. He was treated with rituximab and his immunosuppression regimen was minimized. We review the incidence, pathogenesis, presentation, and management of PTLD in the liver-transplant population. Our case highlights the variation in the presentation of PTLD and the importance of a high index of suspicion among the at-risk group.

Genetic Determinants of Circulating Lipoproteins in Nonalcoholic Fatty Liver Disease.

BACKGROUND: Recent genome-wide association studies have identified 2 genetic polymorphisms in association with nonalcoholic fatty liver disease (NAFLD): patatin-like phospholipase domain containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2), both of which appear to influence the production of very low density lipoprotein (VLDL). The impact of these genetic variations on lipoprotein metabolism in the setting of nonalcoholic steatohepatitis and liver fibrosis are not fully characterized. MATERIALS AND METHODS: We measured comprehensive lipoprotein profiles by nuclear magnetic resonance among 170 serially recruited patients in an NAFLD registry, and determined their relationships with PNPLA3 and TM6SF2 genotypes. RESULTS: In this cohort, 72% patients had at least 1 allele of either PNPLA3 I148M or TM6SF2 E167K, and 30% carried 2 alleles. In multivariate models adjusting for histologic features of nonalcoholic steatohepatitis and liver fibrosis, PNPLA3 I148M is associated with a decrease in VLDL particle size. Both PNPLA3 I148M and TM6SF2 E167K genotypes were associated with increases in the size of low density lipoprotein (LDL) and high density lipoprotein particles, phenotypes considered atheroprotective. When adjusted for both genotypes, NAFLD activity score, in particular the degree of hepatic steatosis was strongly associated with increases in the size of VLDL particles, the concentration of LDL, especially small LDL particles, and a decrease in the size of high density lipoprotein particles, all of which are linked with a proatherogenic phenotype. CONCLUSIONS: PNPLA3 and TM6SF2 are common genetic variants among NAFLD patients and impact lipoprotein profiles in slightly different ways. The interactions between genotypes, hepatic steatosis, and lipoprotein metabolism shed lights on the pathophysiology of NAFLD, and provide opportunities for personalized treatment in the era of emerging NAFLD therapeutics.

Characterizing Normal Bowel Frequency and Consistency in a Representative Sample of Adults in the United States (NHANES).

OBJECTIVES: Our current understanding of normal bowel patterns in the United States (US) is limited. Available studies have included individuals with both normal and abnormal bowel patterns, making it difficult to characterize normal bowel patterns in the US. The current study aims to (1) examine frequency and consistency in individuals with self-reported normal bowel habits and (2) determine demographic factors associated with self-reported normalcy. METHODS: This study used data from adult participants who completed bowel health questions as part of the National Health and Nutrition Examination Survey (NHANES) in 2009-2010 and who reported normal bowel patterns (N=4,775). Data regarding self-perceived bowel health; stool frequency; stool consistency (using the Bristol Stool Form Scale (BSFS)); and demographic factors were analyzed. RESULTS: 95.9% of the sample reported between 3 and 21 BMs per week. Among men, 90% reported a BSFS between 3 and 5, while for women it was 2-6. After controlling for age, the following demographic variables were associated with normalcy: male sex, higher education, higher income, <2 daily medications, and high daily fiber intake. Hispanic ethnicity was significantly associated with abnormal self-reported bowel habits. CONCLUSIONS: This is the first study to evaluate normal bowel frequency and consistency in a representative sample of adults in the US. The current findings bolster the common "3 and 3" metric of normal frequency (3 BMs/day to 3 BMs/week) while also suggesting different criteria for normal consistency for men and women. Finally, this study provides novel information about demographic factors associated with normal frequency and consistency.

Steatohepatitis and liver fibrosis are predicted by the characteristics of very low density lipoprotein in nonalcoholic fatty liver disease.

BACKGROUND & AIMS: A major challenge in the management of nonalcoholic fatty liver disease (NAFLD) is to identify patients with nonalcoholic steatohepatitis (NASH) and early liver fibrosis. The progression of NAFLD is accompanied by distinctive changes in very low density lipoprotein (VLDL), a lipoprotein particle produced exclusively in the liver. Herein, we sought to determine the characteristics of VLDL profiles associated with NASH and liver fibrosis. METHODS: We evaluated VLDL profiles of 128 patients from a single centre NAFLD registry, and examined VLDL size, total and subclass VLDL concentrations in relation to NAFLD activity score (NAS), steatohepatitis and liver fibrosis as determined by liver biopsy. RESULTS: A near linear relationship was observed between mean VLDL particle size and NAFLD activity score (NAS). In multivariate models, VLDL particle size was significantly associated with both NAS and NASH, after adjustment for BMI and diabetes. A decrease in small VLDL particle concentration was associated with more advanced liver fibrosis. In receiver operative characteristic analyses, mean VLDL size performed similarly to cytokeratin 18 in predicting NASH, whereas small VLDL particle concentration had similar performance to NAFLD fibrosis score in predicting stage 2 or above liver fibrosis. CONCLUSIONS: The increase in mean VLDL size in NASH and decrease in small VLDL particle concentration in liver fibrosis likely reflect changes in the number and state of hepatocytes associated with NASH and fibrosis. In addition to its value in risk stratification of cardiovascular diseases, circulating VLDL profile may provide information for the staging of NAFLD disease severity.

Poor Inter-test Reliability Between CK18 Kits as a Biomarker of NASH.

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) affects 15-40% of the general population; 10-20% of those patients have a more severe form of the disease known as nonalcoholic steatohepatitis (NASH). Cytokeratin-18 (CK18), released during apoptosis and one of the most studied biomarkers in NASH, can be measured by a number of commercially available kits. We compared serum measurements of the CK18 M30 from two different kits using the same cohort to evaluate the reliability between two test kits. METHODS: We measured serum levels of CK18 M30 from 185 patients with biopsy-proven NAFLD from a single center from 2009 to 2015, using two different ELISA kits, Test 1 (T1) and Test 2 (T2). Advanced fibrosis was defined as fibrosis stages 3-4 and NASH defined by NAS score ≥ 5. RESULTS: Mean age was 50.2 years (SD 12.6), 61.1% male and 87% White; 49.6% had NASH and 32.2% advanced fibrosis. There was no significant correlation between measurements from the two kits (p = 0.86, r = 0.01). While T2 predicted NASH and advanced fibrosis, T1 did not. The area under ROC curve for the prediction of NASH was 0.631 for T2 versus 0.500 for T1. CONCLUSIONS: Measurements from two different CK18 M30 test kits did not correlate with each other. One kit showed statistically significantly higher levels of CK18 M30 in patients with advanced fibrosis and NASH, while the other kit did not. With the increasing use of CK18 as a biomarker in NASH, it is important to standardize the different kits as it could greatly bias the results.

Surgery for Ulcerative Colitis Is Associated with a High Rate of Readmissions at 30 Days.

BACKGROUND: Currently, the predictors of readmission after colectomy specifically for ulcerative colitis (UC) are poorly investigated. We sought to determine the rates and predictors of 30-day readmissions after colectomy for UC. METHODS: Patients undergoing total proctocolectomy and end ileostomy, abdominal colectomy with end ileostomy, proctocolectomy with ileoanal pouch anastomosis (IPAA) formation and diverting ileostomy, one stage IPAA, or abdominal colectomy with ileorectal anastomosis at a tertiary care center between January 2002 and January 2012 for UC were included. Patients were identified using ICD-9 code 556.x. Each record was manually reviewed. The electronic record system was reviewed for demographic information, medical histories, UC history, medications, and data regarding the admission and discharge. Charts were reviewed for readmissions within 30 days of surgery. Univariate and multivariate analyses were performed using Stata v.13. RESULTS: Two hundred nine patients with UC underwent a colectomy. Forty-three percent had a proctocolectomy with IPAA and diverting ileostomy and 32% had abdominal colectomy with end ileostomy. Seventy-six percent of surgeries were due to failure of medical therapy and 68% of patients were electively admitted for surgery. Thirty-two percent (n = 67/209) of the cohort was unexpectedly readmitted within 30 days. In multivariate model, proctocolectomy with IPAA and diverting ileostomy (odds ratio [OR] = 2.11; 95% CI, 1.06-4.19; P = 0.033) was the only significant predictor of readmission. Hospital length of stay >7 days (OR = 1.82; 95% CI, 0.98-3.41; P = 0.060), presence of limited UC (OR = 2.10; 95% CI, 0.93-4.74; P = 0.074), and steroid before admission (OR = 1.69; 95% CI, 0.90-3.2; P = 0.100) trended toward significance. CONCLUSIONS: Surgery for UC is associated with a high rate of readmission. Further prospective studies are necessary to determine the means to reduce these readmissions.

Missense mutation in APOC3 within the C-terminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerol-rich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen.

Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.

Functional analysis of the missense APOC3 mutation Ala23Thr associated with human hypotriglyceridemia.

We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [(3)H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [(3)H]TAG from C3AT cells was markedly decreased. The lowered [(3)H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL(1). Moreover, [(3)H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [(3)H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [(3)H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL(1) maturation. Furthermore, lumenal [(35)S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL(1) maturation.

Dematin exhibits a natively unfolded core domain and an independently folded headpiece domain.

Dematin is an actin-binding protein originally identified in the junctional complex of the erythrocyte plasma membrane, and is present in many nonerythroid cells. Dematin headpiece knockout mice display a spherical red cell phenotype and develop a compensated anemia. Dematin has two domains: a 315-residue, proline-rich "core" domain and a 68-residue carboxyl-terminal villin-type "headpiece" domain. Expression of full-length dematin in E. coli as a GST recombinant protein results in truncation within a proline, glutamic acid, serine, threonine rich region (PEST). Therefore, we designed a mutant construct that replaces the PEST sequence. The modified dematin has high actin binding activity as determined by actin sedimentation assays. Negative stain electron microscopy demonstrates that the modified dematin also exhibits actin bundling activity like that of native dematin. Circular dichroism (CD) and NMR spectral analysis, however, show little secondary structure in the modified dematin. The lack of secondary structure is also observed in native dematin purified from human red blood cells. (15)N-HSQC NMR spectra of modified dematin indicate that the headpiece domain is fully folded whereas the core region is primarily unfolded. Our finding suggests that the core is natively unfolded and may serve as a scaffold to organize the components of the junctional complex.

The isolated sixth gelsolin repeat and headpiece domain of villin bundle F-actin in the presence of calcium and are linked by a 40-residue unstructured sequence.

Villin is an F-actin regulating, modular protein with a gelsolin-like core and a distinct C-terminal "headpiece" domain. Localized in the microvilli of the absorptive epithelium, villin can bundle F-actin and, at higher calcium concentrations, is capable of a gelsolin-like F-actin severing. The headpiece domain can, in isolation, bind F-actin and is crucial for F-actin bundling by villin. While the three-dimensional structure of the isolated headpiece is known, its conformation in the context of attachment to the villin core remains unexplored. Furthermore, the dynamics of the linkage of the headpiece to the core has not been determined. To address these issues, we employ a 208-residue modular fragment of villin, D6-HP, which consists of the sixth gelsolin-like domain of villin (D6) and the headpiece (HP). We demonstrate that this protein fragment requires calcium for structural stability and, surprisingly, is capable of Ca2+-dependent F-actin bundling, suggesting that D6 contains a cryptic F-actin binding site. NMR resonance assignments and 15N relaxation measurements of D6-HP in 5 mM Ca2+ demonstrate that D6-HP consists of two independent structural domains (D6 and HP) connected by an unfolded 40-residue linker sequence. The headpiece domain in D6-HP retains its structure and interacts with D6 only through the linker sequence without engaging in other interactions. Chemical shift values indicate essentially the same secondary structure elements for D6 in D6-HP as in the highly homologous gelsolin domain 6. Thus, the headpiece domain of villin is structurally and functionally independent of the core domain.

Missense mutations in APOB within the betaalpha1 domain of human APOB-100 result in impaired secretion of ApoB and ApoB-containing lipoproteins in familial hypobetalipoproteinemia.

Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the alpha-helical domain within the N terminus of apoB. Thus, proper folding of the alpha-helical domain of apoB-100 is essential for efficient secretion.

Localization of a critical interface for helical rod formation of bacterial adhesion P-pili.

Pyelonephritic Escherichia coli cause urinary tract infections that involve the kidneys. Initiation of infection is dependent on P-pili expressed on the bacterial surface. In this work, an essential interface for assembly of the helical rod structure of P-pili has been located on the major pilin subunit, PapA. Based on primary sequence alignment, secondary structure analysis, and quaternary structure modeling of the PapA subunit, we predicted the location of a site that is critical for in vivo assembly of the native macromolecular structure of P-pili. A rigid helical rod of PapA subunits comprising most of the pilus length is stabilized by n to n+3 subunit-subunit interactions, and is important for normal function of these pili. Using site-directed mutagenesis, ultrastructural analysis by electron cryomicroscopy, immunocytochemistry, and molecular modeling we show that residues 106-109 (Asn, Gly, Ala, Gly) are essential for assembly of native P-pilus filaments. Mutation of these residues disrupts assembly of the native P-pilus helix. Extended fibrillar structures do still assemble, verifying that n to n+1 subunit-subunit interactions are maintained in the mutant fiber morphology. Observation of this fibrillar morphology in the mutant fiber was predicted by our modeling studies. These mutant P-pili data validate the predictive value of our model for understanding subunit-subunit interactions between PapA monomers. Alteration of the pilus structure from a 7-8 nm helical rod to a 2 nm fibrillar structure may compromise the ability of these bacteria to adhere and remain bound to the host cell, thus providing a possible therapeutic target for antimicrobial drugs.