Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Dec 20;13(6):e0268822.
doi: 10.1128/mbio.02688-22. Epub 2022 Nov 14.

Type IV Pili Are a Critical Virulence Factor in Clinical Isolates of Paenibacillus thiaminolyticus

Christine Hehnly  1 , Aiqin Shi  1 , Paddy Ssentongo  2   3 , Lijun Zhang  4 , Albert Isaacs  5 , Sarah U Morton  6 , Nicholas Streck  7 , Petra Erdmann-Gilmore  8 , Igor Tolstoy  9 , R Reid Townsend  8 , David D Limbrick  10 , Joseph N Paulson  1 , Jessica E Ericson  11 , Michael Y Galperin  9 , Steven J Schiff  12 , James R Broach  1
Affiliations

Type IV Pili Are a Critical Virulence Factor in Clinical Isolates of Paenibacillus thiaminolyticus

Christine Hehnly et al. mBio. .

Abstract

Hydrocephalus, the leading indication for childhood neurosurgery worldwide, is particularly prevalent in low- and middle-income countries. Hydrocephalus preceded by an infection, or postinfectious hydrocephalus, accounts for up to 60% of hydrocephalus in these areas. Since many children with hydrocephalus suffer poor long-term outcomes despite surgical intervention, prevention of hydrocephalus remains paramount. Our previous studies implicated a novel bacterial pathogen, Paenibacillus thiaminolyticus, as a causal agent of neonatal sepsis and postinfectious hydrocephalus in Uganda. Here, we report the isolation of three P. thiaminolyticus strains, Mbale, Mbale2, and Mbale3, from patients with postinfectious hydrocephalus. We constructed complete genome assemblies of the clinical isolates as well as the nonpathogenic P. thiaminolyticus reference strain and performed comparative genomic and proteomic analyses to identify potential virulence factors. All three isolates carry a unique beta-lactamase gene, and two of the three isolates exhibit resistance in culture to the beta-lactam antibiotics penicillin and ampicillin. In addition, a cluster of genes carried on a mobile genetic element that encodes a putative type IV pilus operon is present in all three clinical isolates but absent in the reference strain. CRISPR-mediated deletion of the gene cluster substantially reduced the virulence of the Mbale strain in mice. Comparative proteogenomic analysis identified various additional potential virulence factors likely acquired on mobile genetic elements in the virulent strains. These results provide insight into the emergence of virulence in P. thiaminolyticus and suggest avenues for the diagnosis and treatment of this novel bacterial pathogen. IMPORTANCE Postinfectious hydrocephalus, a devastating sequela of neonatal infection, is associated with increased childhood mortality and morbidity. A novel bacterial pathogen, Paenibacillus thiaminolyticus, is highly associated with postinfectious hydrocephalus in an African cohort. Whole-genome sequencing, RNA sequencing, and proteomics of clinical isolates and a reference strain in combination with CRISPR editing identified type IV pili as a critical virulence factor for P. thiaminolyticus infection. Acquisition of a type IV pilus-encoding mobile genetic element critically contributed to converting a nonpathogenic strain of P. thiaminolyticus into a pathogen capable of causing devastating diseases. Given the widespread presence of type IV pilus in pathogens, the presence of the type IV pilus operon could serve as a diagnostic and therapeutic target in P. thiaminolyticus and related bacteria.

Keywords: Paenibacillus thiaminolyticus; postinfectious hydrocephalus; type IV pilus; virulence factors.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Genomic characterization of clinical isolates of P. thiaminolyticus. (A) Computerized tomography scans from the three infants with postinfectious hydrocephalus from whose CSF the three clinical isolates were recovered. The first two images, from Patient 2033 (clinical strain Mbale) and Patient 2262 (clinical strain Mbale2), were taken prior to surgery and demonstrate loculations and calcified abscess formation. The third image, from Patient 2287 (clinical strain Mbale3), was taken during surgery for hydrocephalus and also shows evidence of extensive brain parenchyma damage. (B) Complete assemblies of the reference strain of P. thiaminolyticus, B-4156T, and three clinical isolates, Mbale, Mbale2, and Mbale3, obtained using long- and short-read sequencing. The B-4156 and Mbale3 genomes each consists of one continuous contig, while the Mbale and Mbale2 genomes each consist of one large contig plus additional contigs (Table 1). (C) Alignment with MAUVE of the clinical isolates’ genomes to that of the B-4156 reference strain identified 12 locally colinear blocks, which are indicated by the different colors. White regions within the colored regions represent regions of low sequence similarity.
FIG 2
FIG 2
Comparisons of the predicted proteomes of the clinical isolates and the reference strain. (A) Venn diagram from OrthoVenn2 analysis comparing the annotations of the B-4156, Mbale, Mbale2, and Mbale3 strains. The 342 orthologous clusters among the clinical isolates that were absent in the nonpathogenic B-4156 are outlined in red. (B) Heat map quantifying the number of predicted orthologous clusters across each isolate. (C) Clinically relevant gene ontology terms from the 342 proteins that were unique to the clinical isolates.
FIG 3
FIG 3
Mobile genetic element identification and annotation. (A) MGEfinder identified regions of genomes that could have been derived from MGEs (red bars) and could account for the regions of low sequence similarity. Genes specified in red were identified by RAST as being encoded in the predicted MGEs and include a lipoprotein, holin, and an arabinose family transcription regulator (AraC TR). A predicted MGE in Mbale and Mbale2 included an operon for a putative type IV pilus, which, although not predicted to be an MGE in Mbale3, was also present in that genome (gray). Regions of phage sequences, identified by PHASTER, are labeled in blue. (B) Bar plot of the predicted functions from the RAST annotation of coding sequences that aligned to the MGEfinder-predicted inserts.
FIG 4
FIG 4
RNA and protein expression of the B-4156 and Mbale strains. (A) Culture growth stages (top) and unsupervised hierarchical clustering (bottom) of the RNA transcript levels of the most variable genes expressed in Mbale and the reference strain of P. thiaminolyticus. Clusters separate primarily according to strains and secondarily on the basis of stage of growth and media (B and C) Comparison of RNA (B) and protein (C) expression between Mbale and B-4156.
FIG 5
FIG 5
Predicted insertion carries the full Gram-positive T4P operon. (A) The T4P operon present in all three clinical isolates and absent in strain B-4156 is located in the predicted mobile genetic element insertion (in strain Mbale, locus tags FLT15_06255 to FLT15_06190). The genes were annotated with RAST or PGAP and/or with hits in the COG and Pfam databases. PilFind (42) was used to identify potential pilins (light brown). White triangles designate hypothetical proteins of unknown function. The asterisk indicates a c-di-GMP-binding site in PilB. (B) Sequence alignment of the N-terminal fragment of PilB from strain Mbale (GenBank accession number NGP58005.1) and truncated protein FLT43_10920 (GenBank number QDM43956.1) from P. thiaminolyticus reference strain B-4156 against experimentally characterized MshEN domains. The top line shows the conserved c-di-GMP-binding site of the MshEN domain, which consists of tandem 24-amino-acid (aa) motifs separated by a 5-aa insert (45). Aligned sequences include MshEN domains of PilB proteins from Clostridioides difficile (47), Clostridium perfringens (46), Pseudomonas aeruginosa (PA3740) (45, 91), and Myxococcus xanthus (MXAN_5788) (92) and of Escherichia coli NfrB (93, 94). The bottom two lines show the sequence and secondary structure (H, α-helix) of the structurally characterized MshEN protein from Vibrio cholerae (VC_0405) (44, 45). Conserved hydrophobic residues are shaded yellow, and conserved Gly residues are shaded green. (C) Expression levels of the pil genes obtained from the transcriptome sequencing (RNA-seq) data.
FIG 6
FIG 6
The T4P is a critical virulence factor in all three clinical isolates. (A) Mouse sepsis model of infection with injection performed postweaning on day 28 of life. (B) Kaplan-Meier survival curve depicting survival of mice following injection of the T4P deletion strain (n = 9; blue), the wild-type Mbale strain (n = 9; red), the reference strain B-4156 (n = 9; orange), or vehicle control (n = 4; black) (P < 0.0001). (C) Amplification of the pilT gene in CSF or bacterial culture (H) from infants with postinfectious hydrocephalus (PIH) due to P. thiaminolyticus or neonatal sepsis (NS) due to P. thiaminolyticus that subsequently led to PIH. Paenibacillus status (Paeni status) was determined by qPCR of the previously defined thiaminase gene and was 100% concordant with pilT positivity (22). L, DNA ladder.
See this image and copyright information in PMC

Similar articles

  • Neonatal Paenibacilliosis: Paenibacillus Infection as a Novel Cause of Sepsis in Term Neonates With High Risk of Sequelae in Uganda.
    Ericson JE, Burgoine K, Kumbakumba E, Ochora M, Hehnly C, Bajunirwe F, Bazira J, Fronterre C, Hagmann C, Kulkarni AV, Kumar MS, Magombe J, Mbabazi-Kabachelor E, Morton SU, Movassagh M, Mugamba J, Mulondo R, Natukwatsa D, Kaaya BN, Olupot-Olupot P, Onen J, Sheldon K, Smith J, Ssentongo P, Ssenyonga P, Warf B, Wegoye E, Zhang L, Kiwanuka J, Paulson JN, Broach JR, Schiff SJ. Ericson JE, et al. Clin Infect Dis. 2023 Sep 11;77(5):768-775. doi: 10.1093/cid/ciad337. Clin Infect Dis. 2023. PMID: 37279589 Free PMC article.
  • Paenibacillus spp infection among infants with postinfectious hydrocephalus in Uganda: an observational case-control study.
    Morton SU, Hehnly C, Burgoine K, Ssentongo P, Ericson JE, Kumar MS, Hagmann C, Fronterre C, Smith J, Movassagh M, Streck N, Bebell LM, Bazira J, Kumbakumba E, Bajunirwe F, Mulondo R, Mbabazi-Kabachelor E, Nsubuga BK, Natukwatsa D, Nalule E, Magombe J, Erickson T, Ngonzi J, Ochora M, Olupot-Olupot P, Onen J, Ssenyonga P, Mugamba J, Warf BC, Kulkarni AV, Lane J, Whalen AJ, Zhang L, Sheldon K, Meier FA, Kiwanuka J, Broach JR, Paulson JN, Schiff SJ. Morton SU, et al. Lancet Microbe. 2023 Aug;4(8):e601-e611. doi: 10.1016/S2666-5247(23)00106-4. Epub 2023 Jun 19. Lancet Microbe. 2023. PMID: 37348522 Free PMC article.
  • Paenibacillus infection with frequent viral coinfection contributes to postinfectious hydrocephalus in Ugandan infants.
    Paulson JN, Williams BL, Hehnly C, Mishra N, Sinnar SA, Zhang L, Ssentongo P, Mbabazi-Kabachelor E, Wijetunge DSS, von Bredow B, Mulondo R, Kiwanuka J, Bajunirwe F, Bazira J, Bebell LM, Burgoine K, Couto-Rodriguez M, Ericson JE, Erickson T, Ferrari M, Gladstone M, Guo C, Haran M, Hornig M, Isaacs AM, Kaaya BN, Kangere SM, Kulkarni AV, Kumbakumba E, Li X, Limbrick DD Jr, Magombe J, Morton SU, Mugamba J, Ng J, Olupot-Olupot P, Onen J, Peterson MR, Roy F, Sheldon K, Townsend R, Weeks AD, Whalen AJ, Quackenbush J, Ssenyonga P, Galperin MY, Almeida M, Atkins H, Warf BC, Lipkin WI, Broach JR, Schiff SJ. Paulson JN, et al. Sci Transl Med. 2020 Sep 30;12(563):eaba0565. doi: 10.1126/scitranslmed.aba0565. Sci Transl Med. 2020. PMID: 32998967 Free PMC article.
  • Pathogenic arsenal of Pseudomonas aeruginosa: an update on virulence factors.
    Veetilvalappil VV, Manuel A, Aranjani JM, Tawale R, Koteshwara A. Veetilvalappil VV, et al. Future Microbiol. 2022 Apr;17:465-481. doi: 10.2217/fmb-2021-0158. Epub 2022 Mar 15. Future Microbiol. 2022. PMID: 35289684 Review.
  • A comprehensive guide to pilus biogenesis in Gram-negative bacteria.
    Hospenthal MK, Costa TRD, Waksman G. Hospenthal MK, et al. Nat Rev Microbiol. 2017 May 12;15(6):365-379. doi: 10.1038/nrmicro.2017.40. Nat Rev Microbiol. 2017. PMID: 28496159 Review.
See all similar articles

Cited by

  • Neonatal Paenibacilliosis: Paenibacillus Infection as a Novel Cause of Sepsis in Term Neonates With High Risk of Sequelae in Uganda.
    Ericson JE, Burgoine K, Kumbakumba E, Ochora M, Hehnly C, Bajunirwe F, Bazira J, Fronterre C, Hagmann C, Kulkarni AV, Kumar MS, Magombe J, Mbabazi-Kabachelor E, Morton SU, Movassagh M, Mugamba J, Mulondo R, Natukwatsa D, Kaaya BN, Olupot-Olupot P, Onen J, Sheldon K, Smith J, Ssentongo P, Ssenyonga P, Warf B, Wegoye E, Zhang L, Kiwanuka J, Paulson JN, Broach JR, Schiff SJ. Ericson JE, et al. Clin Infect Dis. 2023 Sep 11;77(5):768-775. doi: 10.1093/cid/ciad337. Clin Infect Dis. 2023. PMID: 37279589 Free PMC article.
  • Paenibacillus spp infection among infants with postinfectious hydrocephalus in Uganda: an observational case-control study.
    Morton SU, Hehnly C, Burgoine K, Ssentongo P, Ericson JE, Kumar MS, Hagmann C, Fronterre C, Smith J, Movassagh M, Streck N, Bebell LM, Bazira J, Kumbakumba E, Bajunirwe F, Mulondo R, Mbabazi-Kabachelor E, Nsubuga BK, Natukwatsa D, Nalule E, Magombe J, Erickson T, Ngonzi J, Ochora M, Olupot-Olupot P, Onen J, Ssenyonga P, Mugamba J, Warf BC, Kulkarni AV, Lane J, Whalen AJ, Zhang L, Sheldon K, Meier FA, Kiwanuka J, Broach JR, Paulson JN, Schiff SJ. Morton SU, et al. Lancet Microbe. 2023 Aug;4(8):e601-e611. doi: 10.1016/S2666-5247(23)00106-4. Epub 2023 Jun 19. Lancet Microbe. 2023. PMID: 37348522 Free PMC article.

References

    1. Dewan MC, Rattani A, Mekary R, Glancz LJ, Yunusa I, Baticulon RE, Fieggen G, Wellons JC, III, Park KB, Warf BC. 2019. Global hydrocephalus epidemiology and incidence: systematic review and meta-analysis. J Neurosurg 130:1065–1079. doi:10.3171/2017.10.JNS17439. - DOI - PubMed
    1. Isaacs AM, Riva-Cambrin J, Yavin D, Hockley A, Pringsheim TM, Jette N, Lethebe BC, Lowerison M, Dronyk J, Hamilton MG. 2018. Age-specific global epidemiology of hydrocephalus: systematic review, metanalysis and global birth surveillance. PLoS One 13:e0204926. doi:10.1371/journal.pone.0204926. - DOI - PMC - PubMed
    1. Warf BC. 2005. Hydrocephalus in Uganda: the predominance of infectious origin and primary management with endoscopic third ventriculostomy. J Neurosurg 102:1–15. doi:10.3171/ped.2005.102.1.0001. - DOI - PubMed
    1. Warf BC, East African Neurosurgical Research Collaboration . 2010. Pediatric hydrocephalus in East Africa: prevalence, causes, treatments, and strategies for the future. World Neurosurg 73:296–300. doi:10.1016/j.wneu.2010.02.009. - DOI - PubMed
    1. Kulkarni AV, Schiff SJ, Mbabazi-Kabachelor E, Mugamba J, Ssenyonga P, Donnelly R, Levenbach J, Monga V, Peterson M, MacDonald M, Cherukuri V, Warf BC. 2017. Endoscopic treatment versus shunting for infant hydrocephalus in Uganda. N Engl J Med 377:2456–2464. doi:10.1056/NEJMoa1707568. - DOI - PMC - PubMed

Substances

Supplementary concepts

LinkOut - more resources