Drug resistance in Trypanosoma brucei : comparative genomics of melarsoprol-pentamidine cross-resistance and the role of aquaglyceroporin 2 in clinical resistance

Drug resistance in African trypanosomes had already been studied more than 100 years ago, by the pioneering work of Paul Ehrlich. The molecular mechanisms and most genes responsible for drug resistance, however, have not been discovered until recently. New technologies that allow genome-wide comparison were highly successful in identifying many new genes that were linked to drug resistance to all clinical trypanocides. The overall aim of this thesis was to identify and validate candidate genes for drug resistance in Trypanosoma brucei. In Chapter 2, I applied next generation sequencing to find the mutations causing drug resistance in two bloodstream-form T. b. rhodesiense lines that had previously been selected in vitro for resistance against the clinical drugs melarsoprol and pentamidine, respectively. Both cell lines exhibited strong cross-resistance to either drug - a phenomenon first observed over 60 years ago and repeatedly many times - and nowadays the genes involved have been characterized. Comparative genomics revealed the deletion of the known melarsoprol-pentamdine cross-resistance (MPXR) determinants adenosine transporter 1 (TbAT1) in the melarsoprol-selected line and aquaglyceroporin 2 (AQP2) in both selected lines. The pentamidine-selected line had acquired a heterozygous point mutation (G430R) in TbAT1 that rendered the transporter non-functional. The gene TbAT1, encoding the adenosine/adenine permease P2 transporter, has been discovered more than 10 years ago. AQP2 has recently been discovered to play a role in MPXR in a genome-wide RNAi screen. Both transporters mediate the uptake of melarsoprol and pentamidine and, when functionally lost, lead to cross-resistance. AQP2 emerged as the main genetic determinant of MPXR and corresponds to the high-affinity pentamidine transporter. Mutations in AQP2 were found in all analyzed MPXR cell lines selected, either in vitro or in vivo, with arsenicals or pentamidine and from all three T. brucei ssp. (Chapter 3). An additional mutation became fixed in both resistant cell lines; the RNA-binding protein TbUBP1 carried the exact same coding point mutation (R131L). Overexpression of TbUBP1 in T. b. brucei led to a strong growth deficit whereas overexpression of the mutant did not, but intriguingly, those cells became about 2-fold hypersensitive to pentamidine. The physiological function of TbUBP1 and how it affects pentamidine sensitivity remains to be further investigated. TbAT1 and AQP2 are well studied in laboratory cell lines, but knowledge from clinical isolates is scarce. Chapters 4 and 5 investigate drug resistance in clinical isolates. 16 T. brucei ssp. field isolates, 8 stemming from melarsoprol treatment-refractory cases, that had been adapted to axenic in vitro cultivation have been genotyped for TbAT1 and AQP2 to test whether they carry mutations in either transporter and the drug sensitivities have been determined for melarsoprol, pentamidine and diminazene. Indeed, five T. b. gambiense isolates from the Democratic Republic of Congo and one isolate form South Sudan carried a deletion in the AQP2 / AQP3 locus leading to the formation of a chimeric gene between AQP2 and AQP3 and loss of wild-type AQP2. The identified mutant T. b. gambiense isolates were 3- to 5-times less sensitive to melarsoprol and 40- to 50-fold less sensitive to pentamidine compared to reference isolates. Functional expression of the chimeric AQP in a tbaqp2 null background did not restore the drug sensitivity but the introduction of the wild-type AQP2 in one of the resistant T. b. gambiense isolates rendered the cells sensitive to melarsoprol and pentamidine, comparable to fully drug susceptible isolates. This proves that the loss of 'wild-type' AQP2 is the cause of melarsoprol-pentamidine cross-resistance in the T. b. gambiense isolate. Thus AQP2 may serve as a molecular marker for drug resistance in the field

Beyond immune escape:a variant surface glycoprotein causes suramin resistance in Trypanosoma brucei

Suramin is one of the first drugs developed in a medicinal chemistry program (Bayer, 1916), and it is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense. Cellular uptake of suramin occurs by endocytosis, and reverse genetic studies with T. b. brucei have linked downregulation of the endocytic pathway to suramin resistance. Here we show that forward selection for suramin resistance in T. brucei spp. cultures is fast, highly reproducible and linked to antigenic variation. Bloodstream-form trypanosomes are covered by a dense coat of variant surface glycoprotein (VSG), which protects them from their mammalian hosts' immune defenses. Each T. brucei genome contains over 2000 different VSG genes, but only one is expressed at a time. An expression switch to one particular VSG, termed VSGSur , correlated with suramin resistance. Reintroduction of the originally expressed VSG gene in resistant T. brucei restored suramin susceptibility. This is the first report of a link between antigenic variation and drug resistance in African trypanosomes

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporte

Imported Malaria in Children in Industrialized Countries, 1992–2002

Children account for a considerable proportion of cases imported to the United States and Europe

Longitudinal analysis within one hospital in sub-Saharan Africa over 20 years reveals repeated replacements of dominant clones of Klebsiella pneumoniae and stresses the importance to include temporal patterns for vaccine design considerations

Background: Infections caused by multidrug-resistant gram-negative bacteria present a severe threat to global public health. The WHO defines drug-resistant Klebsiella pneumoniae as a priority pathogen for which alternative treatments are needed given the limited treatment options and the rapid acquisition of novel resistance mechanisms by this species. Longitudinal descriptions of genomic epidemiology of Klebsiella pneumoniae can inform management strategies but data from sub-Saharan Africa are lacking. Methods: We present a longitudinal analysis of all invasive K. pneumoniae isolates from a single hospital in Blantyre, Malawi, southern Africa, from 1998 to 2020, combining clinical data with genome sequence analysis of the isolates. Results: We show that after a dramatic increase in the number of infections from 2016 K. pneumoniae becomes hyperendemic, driven by an increase in neonatal infections. Genomic data show repeated waves of clonal expansion of different, often ward-restricted, lineages, suggestive of hospital-associated transmission. We describe temporal trends in resistance and surface antigens, of relevance for vaccine development. Conclusions: Our data highlight a clear need for new interventions to prevent rather than treat K. pneumoniae infections in our setting. Whilst one option may be a vaccine, the majority of cases could be avoided by an increased focus on and investment in infection prevention and control measures, which would reduce all healthcare-associated infections and not just one

Comparative genomics of drug resistance in <i>Trypanosoma brucei rhodesiense</i>

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine

Lactobacillus strains isolated from the vaginal microbiota of healthy women inhibit Prevotella bivia and Gardnerella vaginalis in coculture and cell culture.

International audienceThe purpose of this study was to investigate how human vaginal isolates of Lactobacillus acidophilus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus crispatus inhibit the vaginosis-associated pathogens Gardnerella vaginalis and Prevotella bivia. Results show that all the strains in coculture condition reduced the viability of G. vaginalis and P. bivia, but with differing degrees of efficacy. The treatment of G. vaginalis- and P. bivia-infected cultured human cervix epithelial HeLa cells with L. gasseri strain KS120.1 culture or cell-free culture supernatant (CFCS) results in the killing of the pathogens that are adhering to the cells. The mechanism of the killing activity is not attributable to low pH and the presence of lactic acid alone, but rather to the presence of hydrogen peroxide and proteolytic enzyme-resistant compound(s) present in the CFCSs. In addition, coculture of G. vaginalis or P. bivia with L. gasseri KS120.1 culture or KS120.1 bacteria results in inhibition of the adhesion of the pathogens onto HeLa cells

Vaginal Lactobacillus isolates inhibit uropathogenic Escherichia coli.

The purpose of this study was to investigate the antibacterial activities of Lactobacillus jensenii KS119.1 and KS121.1, and Lactobacillus gasserii KS120.1 and KS124.3 strains isolated from the vaginal microflora of healthy women, against uropathogenic, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strains IH11128 and 7372 involved in recurrent cystitis. We observed that some of the Lactobacillus isolates inhibited the growth and decreased the viability of E. coli IH11128 and 7372. In addition, we observed that adhering Lactobacillus strains inhibited adhesion of E. coli IH11128 onto HeLa cells, and inhibited internalization of E. coli IH11128 within HeLa cells