BamB and outer membrane biogenesis – The Achilles' heel for targeting Klebsiella infections?

KEYWORDS:

• BamB
• Klebsiella
• outer membrane biogenesis

This article is referred to by:

The Klebsiella pneumoniae YfgL (BamB) lipoprotein contributes to outer membrane protein biogenesis, type-1 fimbriae expression, anti-phagocytosis, and in vivo virulence

Over recent decades, Klebsiella pneumoniae has been emerging as the cause of a spectrum of diseases, including both local and systemic infections. In this issue, Hsieh et al. present a comprehensive functional characterization of K. pneumoniae BamB, a non-essential component of the BAM complex involved in biogenesis of outer membrane proteins. In this editorial, their main findings regarding the impact of BamB on K. pneumoniae virulence and the role of BamB as a potential future drug-target are discussed.

Klebsiella pneumoniae is a species of environmental enterobacteriacae that is able to cause a spectrum of community acquired and nosocomial diseases, including infections of the respiratory tract and urinary tract (UTIs), wound infections and sepsis.¹ Over the past decades, emerging syndromes associated with disseminated infection, most notably pyogenic liver abscesses, have been observed in immunocompromised patients with underlying conditions such as diabetes and liver disease.² These are associated with K. pneumoniae faecal carriage,³ and it has been proposed that translocation of bacteria into the bloodstream following colonization of the gastrointestinal tract is the portal to systemic spread and development of disseminated disease.⁴ K. pneumoniae is able to form biofilms on abiotic surfaces including catheders and hospital equipment, and shows high environmental persistence which favors its occurrence as a nosocomial pathogen. Its intrinsically high drug tolerance and ability to acquire drug-resistance lead to the emergence of multidrug-resistant strains, and particularly the emergence of carbapenem resistant strains lead to its inclusion in the Center's for Disease Control and Prevention (CDC) report of antibiotic resistance threats in 2013.⁵

K. pneumoniae virulence factors are not well understood, but mechanisms of host cell interactions and inhibition of efficient clearance by the host immune system seem to be mainly based on surface features, including a capsule, lipopolysaccharide (LPS) and fimbrial adhesins. The capsule protects the pathogen against phagocytosis and innate immune functions as well as desiccation.^6,7 Most clinical isolates express type I and type III fimbriae, which play roles in adhesion to host cells and biofilm formation, respectively.⁸ Type I fimbriae, thin adhesive thread-like surface appendices that extend beyond the capsule, mediate adhesion to epithelial cells via the tip protein FimH, a bacterial lectin. K. pneumoniae FimH binds to terminally exposed Manα(1–3)Manβ(1–4)GlcNAcβ1 trisaccharide in a way that is highly dependent on shear force. While binding is only marginal at low shear force, it is enhanced sevenfold under increased shear, through an allosteric catch bond mechanism.⁹ FimH is found in 90% of K. pneumoniae strains and undergoes pathoadaptive microevolution to favor bacterial persistence within specific host compartments.¹⁰ While it is required for invasion and formation of intracellular bacterial communities (IBCs) by K. pneumoniae in the murine bladder, it is not essential for early colonization.¹¹

Innate immune function, particularly phagocytosis, is key in controlling K. pneumoniae infections and limiting dissemination. - Dexamethasone treatment of Galleria mellonella larvae has been shown to enhance K. pneumoniae virulence.¹² Neutrophils in particular seem to play a key role in K. pneumoniae clearance – it is efficiently cleared when phagocytosed by neutrophils, while the pathogen has been shown to survive within macrophages by avoiding delivery to lysosomes.¹³ However, clearance is limited by the pathogen's natural ability to limit phagocytosis, and both the capsule and LPS confer anti-phagocytic properties in an amoeba model.¹⁴ Thus, it has been suggested that enhancing neutrophil phagocytosis would be an effective approach to target infection.¹⁵ Additional factors help the bacterium to evade mucosal immunity: OmpA confers resistance to antimicrobial peptides,¹⁶ contributes to dampening proinflammatory responses in epithelial cells,¹⁷ along with capsular polysaccharide which also mediates immune evasion, by blocking NFκB signaling.¹⁸ Taken together, these findings make it apparent that targeting individual surface factors may not be sufficient to inhibit virulence, while modulating the biogenesis of surface-associated factors on a larger scale may be an appropriate approach to target K. pneumoniae and treat infection.

The BAM complex is the key machinery responsible for the insertion of β-barrel proteins into the bacterial outer membrane (OM). Because of its link to the biogenesis of surface factors relevant to bacterial virulence in a wide range of pathogens, its structure and function have been subject of investigation for a long time. The BAM complex consists of 5 subunits, termed BamA-E. BamA forms an OM barrel and contains 5 periplasmic POTRA (polypeptide transport-associated) domains, which interact with the periplasmc lipoproteins BamB-E to form a periplasmic ring, with a putative substrate exit portal at the membrane interface. Recent structural studies suggest insertion of OMPs happens by rotation of the periplasmic ring against the membrane barrel domain of BamA.^19,20 While all 5 components are required for optimal activity, only BamA and BamD are essential in E. coli.^21,22 BamB is an accessory protein required for the biogenesis of a subset of OM proteins including porins OmpF, LamB, OmpT ^22,23 but is not required for autotransporter biogenesis,²⁴ highlighting that different subsets of OM proteins have different assembly pathways and requirements. So while BamB is not essential, its absence leads to pleiotropic phenotypes linked to aberrant OM biogenesis, and it plays roles both in membrane permeability and virulence in vivo, which can be genetically segregated.²⁵

Hsieh et al. identified K. pneumoniae BamB (YfgL) as one of the key factors mediating bacterial adherence to epithelial cells following a large scale screen for factors mediating hyperadhesive phenotypes of non-adhesive E. coli.²⁶ Deletion of bamB lead to a 15-fold decrease in K. pneumoniae adherence to retinal, intestinal and lung epithelial cells and consequently decreased invasion, which was complemented by restoring bamB in trans. They showed BamB not to be surface exposed, as expected from its known function as a periplasmic lipoprotein, ruling out a direct role in adhesion. However, deletion of bamB had a pleiotropic effect on the profile of outer membrane proteins, including a decrease in porins OmpA and OmpK35/OmpK36 as well as type I fimbriae. Interestingly, the effect on type I fimbriae expression is not solely due to inhibition of OM biogenesis, but transcription of fimbrial genes is impacted by bamB as well. Notably, fim genes fimA, fimI, fimC, fimF and fimG were all downregulated approximately 4-fold, and fimC/fimD were downregulated approximately 2-fold, along with genes contributing to the PTS system and peptidoglycan synthesis. K. pneumoniae undergoes fimbrial phase variation, which is promoted by an invertible DNA element (“switch”) in the fim promoter. While in wild type K. pneumoniae, this element is in the “off” orientation in all cells under static growth conditions, the ratio is 1:1 between “on” and “off” orientation in cells grown under shaking conditions. In contrast, in a bamB mutant the element remains 100% in the “off” orientation even under shaking conditions, but this effect is not mediated by the PTS system, and a pts mutant is still able to phase switch. How the presence or absence of BamB regulates the fimbrial switch remains to be elucidated. But as a consequence of this change in fim expression, the bamB mutant displayed a 32-fold lower titer of type I fimbriae in a yeast agglutination assay. However, the adhesion defect cannot be attributed to a lack of type I fimbriae alone, since a fim mutant shows a far smaller decrease (∼3-fold) in adherence compared to the bamB mutant. The authors went on to explore the implications of the far-reaching changes in the profile of surface proteins on K. pneumoniae virulence. While wild type cells are not well phagocytosed by neutrophils, as previously described, the lack of bamB leads to enhanced clearance, both by enhancing phagocytosis and by increasing degradation of the pathogen once phagocytosed. Overall, the lack of BamB lead to a significant attenuation in virulence in mice challenged with K. pneumoniae intraperitoneally, and this effect was due to the pleiotropic effect on OM protein biogenesis, rather than the absence of fimbriae alone. While a fim mutant has an LD₅₀ comparable to the wild type, the lethal dose was ∼100 times higher for the bamB mutant. In agreement with its enhanced neutrophil mediated clearance in vitro, the bamB mutant also showed limited dissemination to tissues, and bacterial burdens in liver and spleen were ∼100- and 1000-times lower for the bamB mutant, respectively, while the burden remained unchanged with the fim mutant.

In conclusion, the detailed characterization of bamB in K. pneumoniae by Hsieh et al.,²⁶ including its effect on surface protein biogenesis and virulence, is an important contribution to the field and highlights its importance to the biogenesis of OM porins and fimbriae, not just in terms of membrane biogenesis but also at the transcriptional level. It demonstrates that targeting BamB therapeutically might be a potential strategy to fight K. pneumoniae infections, since its loss causes pleiotropic effects on bacterial physiology but it is non-essential. Consequently, targeted bacteria would be less prone to selective pressure, and less likely to bypass the requirement for functional BamB in a limited number of mutations, since it affects membrane permeability as well as the surface profile which is tightly associated with the requirement for adhesion and immune evasion. Taken together, this means targeting BamB might give us the edge when trying to fight K. pneumoniae infections.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work has been funded by grants BB/M021513/1 and BB/L007916/1 from the Biotechnology and Biological Sciences Research Council (BBSRC).